Exposure to endocrine-disrupting compounds (EDCs) can alter steroid hormone production in vertebrates, sometimes leading to adverse reproductive or developmental effects. Liquid chromatography mass spectrometry methods are the gold standard for analyte confirmation and quantification in biological matrices, but radioimmunoassays (RIAs) are most commonly used for measurement of select steroid hormones in aquatic toxicology studies. Existing methods for steroid quantification often employ derivatization, limiting the range of steroids that can be simultaneously measured in a single process. In the current study, a method for the simultaneous measurement of thirteen endogenous steroids in small sample volumes without derivatization using liquid chromatography atmospheric pressure photoionization tandem mass spectrometry (LC-APPI-MS/MS) was developed. Several physiologically important steroids, including 11-deoxycortisol, 11-ketotestosterone, 17α- and 17β-estradiol, 17α-hydroxyprogesterone, 17,20β-dihydroxyprogesterone, 17,20β,21-trihydroxyprogesterone, androstenedione, cortisol, estriol, estrone, progesterone, and testosterone, were selected for the analysis. The method was validated for application to small volumes of fish plasma and fish holding water. Method detection limits using only 10 µL of plasma ranged from 0.05 to 1.0 ng/mL. As a potential surrogate for plasma steroid measurements, fish holding water was analyzed to measure excreted steroids. Lower limits of quantification when using 0.25 L of water ranged from 0.05 to 1.0 ng/L. The validated method was applied to two different experiments with small fish species exposed to an EDC known to affect steroid synthesis, fadrozole. Concentrations of the 13 steroids were measured in plasma or holding water from the studies. This work demonstrates the potential application of the developed method to measure endogenous steroids for identification of EDCs in aquatic toxicology studies.