Current methods to determine rumen escape (as stability) of rumen protected nutrients(RPNU), such as ruminal in situ evaluation, only estimate the rate at which nutrients leavethe in situ bag (rather than the rumen) and thus can provide only a rough estimate ofactual rumen stability, which is also impacted by actual ruminal RPNU release rate, rateof ruminal passage of the RPNU, as well as impacts of ruminative chewing on the RPNUproduct; all of which can only be estimated. The aim of our study was to use a novel invivo dual liquid phase marker technique to measure actual ruminal stability of three fatcoated RPNU products, and to determine if a common in situ incubation time could matchthe in vivo values determined among products. The three RPNU products were RP ascorbicacid, RP lysine and RP niacin, which were manufactured to contain Co-EDTA, and pulsedosed into the rumen at a single time for each experimental period, concomitant with anequal weight of Cr which was pulse dosed as free Cr-EDTA. The study was a 4 × 4 Latinsquare with 14 d periods using 4 multiparous ruminally cannulated lactating Jersey cows.Rumen instability of the RPNU products was measured as the proportion of the area underthe curve from ruminal in vivo clearance of Co (manufactured into each RPNU product asCo-EDTA) relative to the clearance of Cr (simultaneously rumen dosed as free crystalline Cr-EDTA). The measured rumen payload of the three RPNU products differed despite having thesame fat matrix coating and general characteristics, likely due to differences in the chemicalinteractions of the nutrients with the fat covering, with the in vivo measured payload of RPniacin highest at 656 g/kg, relative to RP lysine at 527 (P<0.05) and to RP ascorbic acid at558 g/kg (P<0.10). In situ incubations of the RPNU products in the same cows, at the sametime, suggested that in situ 30 h dry matter stability was the best predictor of their in vivomeasured rumen stability. This novel in vivo dual liquid phase marker technique can beused to determine the actual rumen stability of any ruminally protected product under anytarget feeding scenario.