High Performance Liquid Chromatography is currently used for the purpose of analytical evaluation of drugs. This is mainly because it allows the separation method both, a qualitative and a quantitative, analysis of high selectivity mixture evaluation and sensitivity. The diploma thesis deals with the issue of purity evaluation of pharmaceutical substance Nalbufin hydrochloride. The aim of the experimental part of the diploma thesis deals with the development and validation of the analytical method for assessing the purity of Nalbufin hydrochloride.An HPLC method was developed on a Nova-Pak C18 column. The mobile phase consisted of two components A and B. MF A composition was as follows: 0.97 g of sodium octane sulfonate was dissolved in 900 mL of water to which were added 100 mL of acetonitrile and 2 mL of triethylamine. Created solution was treated with phosphoric acid to pH 2.5. MF B had the following composition: 0.86g of sodium octanesulfonate was dissolved in 800 mL of water to which 200 mL was added acetonitrile of 2 mL of TEA. The resulting pH was adjusted to pH 2.5 with phosphoric acid. gradient MF had the following composition: From zero minutes from 100% A to 30 min. to 0% A. The 30-60min. 0% A, 60-61 min. with a linear change to 100% A, 61-70 min. equilibrium into the original conditions to 100% A at a flow rate of 1 mL/min. In the following section we evaluated the basic validation parameters: linear dynamic range 0.3 - 4.5 g/mL, we calculated the linear regression equation for Nalbuphine in R2 (0.9999), Oxycodone R2 (0.9999) and Noroxycodone R2 (0.9998). The method gave us detection limits for Nalbuphine 0.069 g/mL. Oxycodone had a detection limit of 0.053 g/mL and Noroxycodone 0.048mg / mL. The limist of quantification in these cases were 0.209 g / mL for Nalbuphine, 0.161 g/mL for Oxycodone and 0.147 g/mL for Noroxycodone. Repeatability for the limit of quantification was also set expressed by the relative standard deviation. For Nalbuphine - RSD = 0.40%, Oxycodone - RSD = 2.39% and Noroxycodone - RSD = 1.25% (RSD 7.0%). The following validation parameter was accuracy. The resulting RSD was 0.44% (RSD 5.0%). The last evaluated parameter was robustness. For pH 2.4, the change value was resolution of 1.5% and repeatability of RSD = 0.85%. The change of resolution value for pH 2.6 was 2.8% and repeatability RSD = 1.29% (max. 5% limit). The second factor observed for the robustness was the temperature change of the column. The arithmetic average was calculated from the individual peak areas and relative standard deviation RSD = 3.45% was evaluated with the change of resolution, which had a value of 3.34%. Thus, we can state that the developed chromatographic method has been verified in the givenvalidation parameters and is suitable for determining the purity of Nalbuphine hydrochloride.