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Baleen whales have few identifiable external indicators of pregnancy state, making it challenging to study essential aspects of their biology and population dynamics. Pregnancy status in other marine mammals has been determined by measuring progesterone concentrations from a variety of sample matrices, but logistical constraints have limited such studies in free-swimming baleen whales. We use an extensive blubber sample archive and associated calving history data to retrospectively identify samples that correspond to pregnant females and develop a progesterone-based pregnancy test for humpback whales. The lowest pregnant blubber progesterone concentration was 54.97 ng g -1 , and the mean for the known-pregnant group was 198.74 ± 180.65 ng g -1 . Conversely, females known to be below the minimum age of sexual maturity (juvenile females) had an overall low mean progesterone concentration (0.59 ± 0.25 ng g -1 ), well below the known-pregnant range. Of the mature females that did not return with a calf ( n = 11), three fell within the known-pregnant range (320.79 ± 209.34 ng g -1 ), while the levels for the remaining eight were two orders of magnitude below the lowest known-pregnant level (1.63 ± 1.15 ng g -1 ). The proportion of females that did not return with a calf but had values similar to known-pregnant females are consistent with rates of calf mortality, but other potential explanations were considered. Our findings support a validated blubber endocrine assignment of pregnancy corroborated with field life history information, a first for any baleen whale species. The progesterone values we measured were similar to those found in different pregnancy states of other cetaceans and support using blubber biopsy samples for assigning pregnancy in humpback whales. This method can be applied to existing archives or new samples to better study life history and population demography broadly across species and populations.