Novel peptide-based drugs have recently gained high popularity, especially among athletes seeking ways to enhance their performance. Although the World Anti-Doping Agency (WADA) has banned the use of any nonapproved for human therapeutic use pharmacological substances in Sports, a huge variety of such peptides with potential performance-enhancing properties are available for sale in the black market and in illegal online websites. The difficulty of determination of these molecules in biological fluids depending on their low concentrations and their similarity to endogenous compounds has boosted their use not only among athletes, but also in the amateurs’ world. The goal of this study was to perform the mass spectrometry characterization of a novel s597 peptide drug purchased via an online store. The study was carried out using nanoscale liquid chromatography/quadrupole Orbitrap mass spectrometry with accurate mass determination and sequence analysis for both intact drug and after its trypsinolysis. The purchased drug was found to be a peptide with 31 amino acid residues, intra-chain disulfide bond and modified by C-terminal amide and N-terminal acetyl groups. The proposed sequence was consistent with s597 peptide, designed to be used as insulin receptor ligand mimetic.