Rationale: Wildlife scientists are quantifying steroid hormones in a growing number of tissue types and employing novel methods which must undergo validation before application. This study tested the accuracy and precision of liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for use on blubber samples from short-finned pilot whales ( Globicephala macrorhynchus). We expanded upon a method for corticosteroid quantification by increasing the number of analytes and optimizing internal standards application. Methods: We optimized a method for the quantification of seven steroid hormones using LC-MS/MS with a C18 column. We assessed the accuracy and precision of this updated C18 method and an existing Biphenyl method for use with short-finned pilot whale blubber tissue by conducting a spike-recovery experiment and calculating percent recovery and relative standard deviation (RSD) for each analyte. To explore the potential for running this method more cost-effectively with fewer matched internal standards (IS), we compared the performance of multiple internal standards for each analyte. Results: The C18 method produced reliable quantitation for the seven target adrenal steroids. The measurement of all 11 adrenal and gonadal analytes was both accurate and precise, with percent recoveries between 82 % to 110 % and RSDs below 10 %. IS comparisons showed 10 of 11 analytes could be calculated accurately and precisely with at least one of the IS substitutes. Though many internal standard substitutions met percent recovery and RSD requirements, some of these substitutions significantly altered the analyte concentrations calculated. Discussion: The methods developed and tested in this study provide reliable detection and quantification of 11 steroid hormones, including DHEA, which has not been previously quantified in blubber. These methods can be used for more comprehensive assessments of adrenal and gonadal steroid hormones from whales. Laboratories can reduce costs through IS substitution but should consider how these substitutions might affect results.