A comparison was carried out between a simple method of High performance Liquid Chromatography (HPLC), Gas Chromatography (GC), enzymatic and colorimetric methods for determination of cholesterol levels. Linearity, accuracy, precision and detection limits were evaluated for HPLC and the other analytical methods. The results revealed that the HPLC method recorded the best linearity (correlation coefficient, r = 0.99992), followed by the GC (r = 0.99874), the enzymatic (r =0.99469) and the colorimetric method (r = 0.99316). The accuracy of the obtained data were calculated based on recovery of the cholesterol added to butter at three concentrations, (75, 150 and 225 mg/100g). At the low concentration (75 mg/100g), the best recovery was estimated by HPLC. In contrast, the GC and the enzymatic methods showed greater recovery at the other two concentrations when compared with HPLC. However, the recovery which has been obtained by colorimetric method was above the limit, for the three concentrations. The precision results showed no significant differences when the cholesterol was determined within 4 days by HPLC, GC and colorimetric methods. However, when it was determined by the enzymatic method the precision results recorded significant differences within 2 days. The HPLC method was very sensitive to low concentration of cholesterol (detection limit 24 µ/µL) followed by the GC (detection limit 49 µ/µL), the colorimetric (detection limit 9 µg/µL) and the enzymatic method (detection limit 11 µg/µL). The HPLC method has been successfully applied for quantitative assay of cholesterol in a variety of animal food sources.