Olive oil is a food that has an impressive chemical composition and it is especially interesting in the context of polyphenols, whose antioxidant activity in the body develops even at low concentrations. One of the most important goals in the analysis of polyphenols in olive oil is to obtain the most accurate relationship between the measured (actual) concentration of the component in the sample with the concentration that ultimately reaches targeted sites in the cells and affects biological processes in them. HPLC and NMR are two standardized methods for determining the concentration of certain phenolic compounds in olive oil extracts. The concentration of oleocanthal and oleacein was determined in extracts prepared using acetonitrile and cyclohexane and analysed by NMR spectroscopy, while the concentration of hydroxytyrosol, tyrosol, pinoresinol, cinnamic acid and apigenin was determined in extracts prepared using methanol and hexane using HPLC-DAD spectroscopy.The polyphenolic composition of six olive oils of autochthonous Croatian olive varieties was tested: Rosulja (Oleum Viride), Buža (Brist), Bjelica (Brist and Oleum Viride), Vodnjanska crnica (Oleum Viride) and Multi-varietal oil Istra. The content of individual phenolic compounds differed depending on the olive variety. In most of the examined cultivars, the oleacein concentration was higher than the oleocanthal, which is in line with the previous data available so far on Croatian cultivars, while the opposite ratio of these two phenols was found in olive oils cultivated in other geographical regions. The tested olive oils can be considered a good source of polyphenolic components and indicator of the exceptional quality of these oils from the territory of the Republic of Croatia. After analysis, both methods of extractions were compared in order to determine the optimal procedure for preparing the extracts for further biological research. The procedure was performed so that both extracts were run on both HPLC and NMR (regardless of which extraction method was used). However, comparisons of these two methods for extraction did not give satisfactory results.