The aim of the study was to develop a method for the determination of 2-methoxyhydroxybenzene in biological material. TLC, UV spectrophotometry, HPLC and GC-MS were used in the experiments. The use of a mixture of ethyl acetate-acetone (7:3 by volume) for the isolation of 2-methoxyhydroxybenzene from biological material is justified. Optimal isolation conditions are established. Purification of the substance was carried out by extraction and chromatography in sorbent column (KCC-3) 80/120 μm. For preliminary identification, TLC was used on Sorbfil PTSX-AF-A-UV plates. Confirmation of identification was carried out by the UV spectrum in ethanol by HPLC with the retention time in a 250×4.6 mm column «SunFire C18» (mobile phase acetonitrile-0.025 M potassium dihydrogen phosphate solution 6: 4). Confirmation identification and quantification was performed by GC-MS using a fixed phase capillary column of 5% phenyl-95% methyl polysiloxane after derivatization of the analyte with N-trimethylsilyl-N-methyl trifluoroacetamide (heating for 30 min at a temperature of 60 °C). Ions 45, 58, 73, 91, 107, 136, 151, 166, 181, 196 m/z are present in the mass spectrum of the derivative. The validation of the methodology for the determination of 2-methoxyhydroxybenzene in biomaterial based on the application of the GC-MS method was carried out. The compliance of the methodology with the criteria of linearity, selectivity, correctness, precision and stability is established. The detection limit and the limit of quantification are 8 and 15 μg per 100 g of biomaterial, respectively.