A direct saponification and normal-phase high-performance liquid chromatography (NP-HPLC) procedure was developed for simultaneous determination of cholesterol and retinol in meat. A normal-phase silica column fitted with diode array detection at 208 nm for cholesterol and fluorescence detection (lambda-excitation 344 nm/lambda-emission 472 nm) for retinol, with mobile phase consisting of 2% (v/v) 2-propanol in n-hexane was used. Cholesterol was eluted at 10 min and retinol at 12.66 min. High linearity (R (2) > 0.9996 for both compounds) in calibration range was obtained. The LOD and LOQ values show the high sensitivity of the developed methodology for simultaneous determination of cholesterol and retinol in meat. Recovery results obtained in this study (98.67-102.14% for cholesterol and 91.72-98.27% for retinol) were between AOAC recommendations to validated method and were comparable to most recent studies in precision and accuracy. In addition, the present method showed high repeatability and reproducibility. As a general conclusion, the results indicate that the direct saponification, extraction, and HPLC analysis is an adequate method for cholesterol and retinol analysis in meat samples.