Despite important applications in anti-doping testing and endocrinology, the differentiation and characterization of steroid structures remain an analytical challenge. A key difficulty is the presence of isomeric species with only subtle structural differences. Here, we analyze two sets of isomeric steroids: the corticosteroids, aldosterone and cortisone, as well as 11β-, 17α-, and 21-hydroxyprogesterone. When probed with ion mobility, the analytes exhibit little variation in collision cross section values on a Synapt G2-S, making their separation from mixtures challenging. Ion mobility also offers limited information about their gas-phase structures. Cryogenic gas-phase infrared spectroscopy provides an additional dimension for identification and distinguishes between the isomers, enabling definitive structural elucidation. In combination with density functional theory, we present experimental data on the preferred protonation of the C3 carbonyl group. Notably, for aldosterone, an unusual bicyclic structure was identified. Overall, this work showcases the value of gas-phase infrared spectroscopy for steroid differentiation and identification, potentially contributing to anti-doping measures and diagnostics for adrenal and congenital disorders.