Lipophilic vitamins are organic compounds which are essential for a variety of physiological functions in the human body, such as vision (vitamin A), calcium and phosphate homeostasis (vitamin D), antioxidative protection in cell membranes (vitamin E) and blood coagulation (vitamin K). As lipophilic vitamins, with the exception of vitamin D3, can not be obtained by endogenous synthesis, food or vitamin supplements are their main source. A public opinion poll of the Ministry of Health showed that vitamin preparations are the second most frequently used nutritional supplements, so quality control of these preparations is extremely important, especially because of the risk of toxicity from excessive intake of vitamins A and D. Therefore, the main objective of the thesis was to evaluate the content and the stability of lipophilic vitamins in selected medicines and nutrition supplements at two different storage temperatures (25°C and 40°C). For this purpose, a rapid and simple analytical method for simultaneous quantification of five lipophilic vitamins (in most commonly used forms – A-palmitate, D3, K1, E-acetate and provitamin A – β-carotene) and coenzyme Q10 in liquid and solid preparations was developed. The method involves simple and fast extraction procedures: dilution with the mixture of tetrahydrofuran, acetonitrile and water (50/45/5, v/v/v) containing butylated hydroxytoluene for liquid preparations and extraction with n-hexane and evaporation of organic extracts under nitrogen at 40°C for solid preparations. The samples were analyzed by high performance liquid chromatography coupled with UV detector. The method was successfully validated according to the ICH guidelines in terms of selectivity, linearity (R2 > 0,999), adequate accuracy (96 – 102%), precision (RSD < 2%), robustness and extraction efficiency (93–108%) for all anaytes. The adequacy of the method was confirmed by testing 12 vitamin preparations containing one or more lipophilic vitamins and coenzyme Q10. The content of vitamins in the majority of tested preparations was higher than declared on the label. The biggest deviation from declared content was observed in two nutritional supplements. The three-month stability study after opening has revealed that lipophilic vitamins are more unstable at 40°C. At both storage temperatures, liquid preparations were more unstable compared to solid preparations. At 25°C, only in liquid preparations significant decline in the content was observed, where A-palmitate was found to be at least stable.