Vitamins are the essential nutrients in the human organism involved in many physiological processes, contributing to both, the health and the good look. There are water-soluble and fat-soluble vitamins. This thesis is based on water-soluble vitamins since biotin is part of vitamin B complex which are water-soluble. Due to the inability of the intake of recommended dietary allowance of vitamins via food, and the potential of resolving the health issues, people are increasingly reaching out for the food supplements. There are multivitamin preparations on the market that are not subject to strict quality control, many of which containing less than the declared amount of supplements. The aim of this study is to confirm the presence of the biotin in the monovitamin food supplement by mass spectrometry, to optimize the conditions of its chromatographic analysis and to determine the biotin content in vitamin preparation. Liquid chromatography coupled with electrospray ionization and mass spectrometer (HPLC-ESI-MS/MS) was used for analysis. The content and mobile phase flow rate were selected first. Isocratic elution with mobile phase consisting of water:methanol:formic acid = 70:30:0.1 (v/v/v), at the flow rate of 0.8 ml/min was used followed by the fragmentation and MSn analysis, resulting in the identification of the characteristic fragments and the structural identification of biotin in the sample. No chemical reference standard of biotin was available, hence another monovitamin food supplement was used as reference substance. The same reference substance was also used for quantification. The results show that biotin is present in the analysed vitamin food supplement, which were purchased through internet shopping. The defined amount of biotin in the sample is 8.4 mg that approximately corresponds to the sample declaration of 10 mg of biotin.