A selective and sensitive analytical method for the determination of selected catechins (catechin, epicatechin, gallocatechin, epigallocatechin) and pyrogallol in biological matrices by HPLC-MS/MS was developed. The utilized sample preparation technique was a two-stage liquid–liquid extraction using ethyl acetate. The HPLC-system was equipped with a Phenomenex Luna Pentafluorophenyl Column (150 × 2 mm, 5 µm) and operated with an acetonitrile–water gradient as a mobile phase system. Detection was performed with a 3200 Q Trap mass spectrometer. For analysis the mass spectrometer was used in the MRM-mode with negative ionization. The method validation was performed with serum as matrix. The selectivity of the method as well as the linearity of calibration was successfully proven for all analytes. The limits of quantification were between 5.3 and 11.2 ng/mL and the recovery rates were above 50 % for all analytes. Results from the samples of three deer poisoning cases demonstrated that the developed HPLC-MS/MS method is applicable to real biological samples.