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 1H 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).