© 2019 Society for Marine Mammalogy Hormonal biomarkers are useful indicators of mammalian reproductive and metabolic states. The present study validated and applied the use of progesterone and cortisol blubber assays for studies of blue whales from the Gulf of California, Mexico. In a validation study for pregnancy detection, blubber progesterone concentrations were correlated with pregnancy status for four female blue whales: three resighted with a calf the year following sampling and the fourth stranded with a fetus. The progesterone concentrations were significantly higher than those measured in juvenile whales (n = 3). In the application study, blubber samples from blue whales (51 noncalf females, 2 female calves, 48 noncalf males, and 1 male calf) with known sighting histories were analyzed. Putative pregnant females had elevated progesterone concentrations. Cortisol concentrations did not differ between male and female blue whales, or among females in different reproductive classes. After correcting for uncertain ages, hence maturity status, the pregnancy rate of noncalf females was 33.4% (95% CI 32.2%–34.3%). Although interpretation of hormone biomarkers must consider all physiological states that may influence progesterone concentrations, these results demonstrate the utility of pairing hormone biomarkers with sighting histories to help assess environmental or anthropogenic impacts on reproduction in blue whales.