AbstractExtensive evidence of the deleterious biological effects of oxidized 5‐cholesten‐3β‐ol (cholesterol) derivatives has led to great interest in their detection. We observed that known oxidized cholesterol derivatives can be rapidly quantitated by combining reversed‐phase high‐performance liquid chromatography (HPLC) with ultraviolet (UV) absorption and evaporative laser light‐scattering (ELSD) detection. Using a 20 × 0.46 cm C18 HPLC column and methanol/acetonitrile (60:40, vol/vol) as the mobile phase at 1.0 mL/min, 10 species of oxidized cholesterol derivative were measured by UV (205, 234, and 280 nm) while 5‐cholestan‐5α,6α‐epoxy‐3β‐ol (5α‐epoxycholesterol), 5‐cholestan‐5β,6β‐epoxy‐3β‐ol (5β‐epoxycholesterol), and 5‐cholestan‐3β,5α,6β‐triol (cholestanetriol) were detected by only ELSD. The minimal limits of detection ranged from 100 to 500 ng depending on sterol and detector. The response was linear in the range 0–1000 or 0–2000 ng depending on detector. These oxidized cholesterol derivatives were also identified by HPLC/mass spectrometry analysis combined with UV detector. Heated tallow contained cholestanetriol, 5‐cholesten‐3β,7α‐diol (7α‐hydroxycholesterol), 5‐cholesten‐3β,7β‐diol (7β‐hydroxycholesterol), 5‐cholesten‐3β‐ol‐7‐one (7‐ketocholesterol), 5α‐ and 5β‐epoxycholesterols under the developed analysis condition. Photooxidized cholesterol had cholestanetriol, 7α‐ and 7β‐hydroxycholesterols and 3,5‐cholestadien‐7‐one. On the other hand, 7α‐ and 7β‐hydroxycholesterols, 7‐ketocholesterol, 5α‐ and 5β‐epoxycholesterols and 3,5‐cholestadien‐7‐one were observed in copper‐oxidized low‐density lipoprotein. Thus, this developed HPLC analysis method could be applied to identification of oxidized cholesterol derivatives in food and biological specimen.