In 2020, the confirmation of the non-endogenous origin of several pseudo-endogenous steroids by means of isotope ratio mass spectrometry (IRMS) was recommended by the World Anti-Doping Agency (WADA), in addition to previously established target analytes for IRMS in sports drug testing. To date, however, IRMS-based methods validated in accordance with current WADA regulations have not been available. Therefore, the aim of this research project was the development and validation of a method to determine the carbon isotope ratios (CIR) of all newly considered pseudo-endogenous steroids, encompassing the anabolic androgenic steroids comprising a 1-ene-core structure (5α-androst-1-ene-3β,17β-diol, 5α-androst-1-ene-3,17-dione [1AD], 17β-hydroxy-5α-androst-1-en-3-one, 3α-hydroxy-5α-androst-1-ene-17-one [1AND], and 3β-hydroxy-5α-androst-1-ene-17-one [1EpiAND]), as well as steroids referred to as hormone and metabolic modulators (androsta-1,4,6-triene-3,17-dione [TRD] and its main metabolite 17β-hydroxy-androsta-1,4,6-triene-3-one) and 6α- and 6β-hydroxy-androst-4-ene-3,17-dione. With peak purity of target analytes being critical for IRMS analyses, a twofold high-performance liquid chromatography (HPLC)-based sample purification was employed, with all analytes being acetylated between the first and second HPLC fractionation. Using established gas chromatography/combustion/IRMS instrumentation, limits of quantification were estimated at 10 ng/ml for a 20 ml urine aliquot for all analytes, except for 1AND (20 ng/ml), and combined measurement uncertainties were estimated between 0.4‰ and 0.9‰. For proof-of-concept, samples collected after the single oral administration of a nutritional supplement containing 1AD and 1EpiAND were analyzed as well as existing excretion study urine samples obtained after the administration of 4-androstenedione and TRD. Based on the obtained results, the developed method was considered to be fit-for-purpose.