Background The purpose of the study is to develop a suitable analytical method in order to establish appropriate conditions for isolation and assay of the dominant compounds in extracts of Capsicum annuum. Results The studies are performed with standard substances to establish the HPLC conditions for complete separation of capsaicin and dihydrocapsaicin, the two major components of interest. Because of their prevalent apolar features, reverse phase chromatographic version was approached. Systematic studies on different eluents revealed the 65% methanol–35% water mixture as a proper mobile phase providing a complete separation of the components according to analytical claims. Conclusions The results may be of interest to develop a specific analytical procedure with advanced specificity for quantitative assay of capsaicin and dihydrocapsaicin in pharmaceutical products and in foods.