Abstract Venlafaxine, one of the most commonly prescribed antidepressants, very often is in a displayed medicinal therapy together with the selective β1 receptor blocker metoprolol. In this regard, the research and development of reliable analytical methods for self-determination, as well as mixtures in chemical and biological samples is essential to provide fast and reliable information to the institutions of the type of substance concentration, the presence of metabolites and other chemical characteristics. Research teams in this field work in several directions – creating analytical HPLC methods for identification, purity and assay tests, their validation and testing them to extend their application to more than one substance and determination in respect of the time. In this connection there were optimized chromatographic and analytical parameters such as retention time, resolution, column efficiency as number of theoretical plates, capacity factors, specificity, repeatability, LOD, LOQ, linearity and system suitability test.