Obtaining longitudinal endocrinological data from free-ranging animals remains challenging. Steroid hormones can be extracted sequentially from non-invasively sampled biologically inert keratinous tissues, such as feathers, nails, hair and whiskers. However, uncertainty regarding the type and levels of steroids incorporated into such tissues complicates their utility in wildlife studies. Here, we developed a novel, comprehensive method to analyze fourteen C19 and fourteen C21 steroids deposited chronologically along the length of seal whiskers in a single, 6-minute chromatographic step, using ultra-performance convergence chromatography-tandem mass spectrometry. The limits of detection and quantification ranged from 0.01 to 2 ng/mL and from 0.1 to 10 ng/mL, respectively. The accuracy and precision were within acceptable limits for steroids at concentrations ≥2 ng/mL. The recovery (mean = 107.5% at 200 ng/mL), matrix effect and process efficiency of steroids evaluated, using blanked whisker matrix samples, were acceptable. The method was applied to the analysis of steroid hormone levels in adult female whisker segments obtained from southern elephant seals (Mirounga leonina), n = 10, and two fur seal species, Antarctic fur seals (Arctocephalus gazella; n = 5) and subantarctic fur seals (Arctocephalus tropicalis; n = 5), sampled between 2012 and 2017. In the whisker subsamples analyzed (n = 71), the median concentration of steroid hormones detected above the LOQ ranged from 2.0 to 273.7 pg/mg. This was the first extraction of multiple C19 and C21 steroids, including their C11-oxy metabolites, from the whiskers of mammals. Measuring hormones sequentially along the whisker lengths can contribute to our understanding of the impact of stress associated with environmental/climate changes that affect the health, survival of organisms, as well as to delineate the reproductive cycles of free-living mammals with cryptic life stages.