A simple, fast, and sensitive method for the extraction through direct saponification, separation, quantification, and identification of 12 cholesterol oxidation products (COPs) and cholesterol in a single isocratic, normal-phase, high-performance liquid chromatography (HPLC) was developed. Three detectors were compared for determination of COPs and cholesterol in fish samples: refractive index (RI), ultraviolet (UV), and atmospheric pressure chemical ionization mass spectrometry (APCI-MS). The results did not show significant differences (p > 0.05) between the concentration of the cholesterol oxides and cholesterol obtained with these detectors. The present study demonstrated the presence of 19-hydroxycholesterol, 22"R"-hydroxycholesterol, 22"S"-hydroxycholesterol, 24"S"-hydroxycholesterol, and 25"R"-hydroxycholesterol for the first time in fish samples.