As a result of studying 43 endogenous steroids, we obtained their full mass spectra, retention times and characteristic ions. When constructing the calibration dependencies, we compared several matrices and showed their advantages and disadvantages. The developed technique was used for more than three years for the real samples analysis. On the base of the obtained data, for the first time we set reference limits for Russian athletes, and a form of the data is consistent with the literature, and the maximal concentration values set for the male and female population of Russian athletes vary significantly from the limits set by WADA. New significant markers of taking prohibited drugs by using Statistica were founded. This approach discovers a fundamentally new method for studying steroid profile, allowing estimation of changes rather in the ratio of steroid metabolites than in the concentrations of steroids themselves. Moreover, this approach can be used in future not only for determining the administration of prohibited drugs, but also for looking for significant markers in the determination of various diseases that lead to changes in steroid hormones.