Determination of rumen escape of rumen protected nutrients is needed to accurately assess the amount of nutrients that can be absorbed and utilised from the intestinal tract of dairy cows. This assessment allows more precise feeding of specific nutrients, thereby increasing metabolic efficiency and reducing production of animal wastes. The currently used method of choice to determine the rumen escape of rumen protected nutrients is ruminal in situ evaluation, which cannot measure actual rumen escape, as the experimentalist can only estimate the rate at which a rumen protected product (RPP) will exit the rumen. The two-part objective of the study was to use an in vivo dual liquid phase marker system to determine ruminal stability of RPP and to determine the stability of the three RPP, as well as determine the best in sacco incubation time to match the determined stability. Four multiparous, ruminally cannulated, lactating Jersey cows [body weight 384 kg ± 28.0 kg, milk yield 24 ± 4.0 L, days in milk 69 ± 42 d, parity 4.5 ± 1.29 (mean ± standard deviation)] were used in a 4 x 4 Latin square design. Cows were assigned to one of four groups with one group being the control group and the other three each receiving a different RPP. The study was composed of four 14 d periods with a 10 d adaptation period before the start of the study to allow the cows to adjust to the individual stalls, diets and conditions. Days 1-6 of each period were for recovery/ rest, days 7-8 for an in sacco measurement to determine the stability of the RPP, days 8-11 for pH logging, and days 11-14 to determine the in vivo stability of the RPP. Cows were fed ad libitum a common total mixed ration (TMR) composed of chopped lucerne hay, maize stover, maize meal, soybean oilcake, hominy chop, molasses, urea, rumen inert fat and a vitamin/mineral premix containing 180 g/kg crude protein, 317 g/kg neutral detergent fibre and 213 g/kg starch on a dry matter basis. Cows were fed twice daily at 2 kg above their daily voluntary feed intake level and kept in individual stalls of 6 x 6 m with wood chips on the floor as bedding. The three RPP were: RP Ascorbic Acid (A), RP Lysine (L) and RP Niacin (N). The RP A and RP N were both composed of 623 g/kg nutrient (Ascorbic acid/ Niacin respectively), 89 g/kg Co-EDTA and 288 g/kg fat matrix, with a measured specific gravity of 1.21. The RP Lysine was composed of 518.7 g/kg Lysine, 86.5 g/kg of Co- EDTA and 394.8 g/kg fat matrix, with a measured specific gravity of 1.21. The fat matrix used in all the RPP’s was the same. The method used in this study aimed to create an accurate quantitative value of true ruminal stability, which traditional methods lack. Stability of the RPP was measured as the proportion of the area under the curve from the ruminal clearance of Co (included in the RPP as Co-EDTA) relative to the clearance of the Cr (as free Cr-EDTA). In sacco measures consisted of insertion of six Dacron bags into each treatment cow (i.e. A, L, N), with each Dacron bag containing 5 g of the relevant product and each cow receiving a different product. Two bags were collected after 12, 24 and 30 h of incubation and then weighed back to determine the stability of the RPP as well as disappearance of the RPP over the 30 h period. Ruminal pH logging occurred directly after and the pH loggers were left in the rumens for 48 h to measure pH every 10 min in each cow. During in vivo measurements each cow was dosed simultaneously with 150 g of the relevant RPP (calculated to contain 15g Co-EDTA) as well as 16.679 g of Cr-EDTA (Control group was dosed with 16.679 g Cr-EDTA and 15 g Co-EDTA) calculated to deliver 2.4 g of Co and 2.4 g of Cr respectively, into the rumen of each cow. Pre-dosing rumen fluid samples were collected and samples were then collected, starting one hour post dosing, every 2 h through 25 h post-dosing, then every 4 h until 49 h post dosing, and thereafter every 6 h until 73 h post dosing. These samples were analyzed for Co, Cr and pH. Samples were also collected every 6 h throughout the 74 h test period for nitrogen ammonia and volatile fatty acid analysis. Rumen pH was within normal ranges and showed normal diurnal variations during sampling. Ruminal pH was unaffected by in vivo treatment and averaged 5.88, with a diurnal variation between 5.65 and 6.40. Animal performance was unaffected by treatment with average milk production of 24.6 L/ day, milk fat of 41.8 g/ kg and milk crude protein of 35.6 g/ kg. The rumen stability of the RPP differed, despite having the same fat matrix, presumably due to differences in the chemical interactions of the nutrients with the fat cover; for example lysine is known to be more reactive. The rumen stability of RP Niacin was the highest (p = 0.06) at 66.7% relative to RP Lysine at 55.0%, with RP Ascorbic acid at 58.7%. In sacco incubations of the RPP showed variation in results. This in vivo method can be utilised to quantitate rumen stability of RPP, although it can not indicate the most appropriate rumen in sacco incubation time to reflect that measurement.