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Whey protein-based supplements are one of the most consumed products to improve performance, and they are, generally, recognized as being safe. However, recent studies indicated different types of adulterations in these products, including the presence of diuretics and stimulants, allowing the possibility of contamination by other substances with pharmacological activities, such as anabolic agents. In this sense, the aim of this study was to develop and validate a method for the screening of 46 anabolic agents in whey protein. The analytical method consisted of sample preparation using liquid-liquid extraction, followed by analytes derivatization and, finally, analysis by gas chromatography coupled to triple quadrupole mass spectrometry in two different experiments: multiple reaction monitoring and full scan. After validation, the method proved to be specific, accurate, and precise; no carry over effect was observed, and the limit of detection values varied between 0.5 and 100 ng g−1. The analysis of 44 commercial samples did not detect the presence of any anabolic agent. However, full scan analysis detected caffeine in 14 samples and bisphenol A in one of them. Therefore, these results indicate that the analyzed samples are safe concerning the presence of the evaluated anabolic agents, but it does not mean that they are free from the presence of other substances with pharmacological activities which are not declared on label. [ABSTRACT FROM AUTHOR]