The aim of this study was to compare two methods for determining the fatty acid methyl ester content of biodiesel by applying high-performance liquid chromatography (HPLC-UV) and the reference method based on gas chromatography (GC). The samples of biodiesel were also analyzed by H-1 NMR. External standards were used for the quantification of fatty acid methyl esters by HPLC-UV. The analytical curves of the methyl oleate, methyl linoleate, and methyl linolenate standards showed good linearity (coefficient of determination >= 0.995). Cochran's test was used to assess the homoscedasticity of the analytical curves. The limits of detection and quantitation were found: 0.0018% mass and 0.0054% mass for methyl oleate, 0.0002% mass and 0.0007% mass for methyl linoleate, and 0.0001% mass and 0.0004% mass for methyl linolenate. The accuracy of the HPLC-UV method was assessed by determining recovery (%), which resulted on values between 81.7 +/- 0.2 and 110.9 +/- 0.1. Precision was assessed by determining repeatability (%), which was found to be between 0.2 and 1.3. The proposed HPLC-UV method proved efficient in determining the fatty acid methyl ester content of biodiesel and the paired t-test showed it to correlate well with the reference method (GC).