Background: Oxysterols are hydroxy, keto or epoxy oxidation products of cholesterol formed by enzymatic action or auto-oxidation. Oxysterols have been shown to have several biological activities including regulation of cholesterol homeostasis. Oxysterols have also been suggested to be informative biomarkers of oxidative stress. The goal of this project is to develop and validate a high performance liquid chromatography-mass spectrometry (HPLC-MS) method for determination of major oxysterols in human plasma. Methods: A reverse phase, high performance liquid chromatography-mass spectrometry (HPLC-MS) method was developed. Cold saponification with ethanolic sodium hydroxide was used to extract total oxysterols from plasma. The effect of sample preparation steps on the stability of oxysterols was characterized. A blank plasma matrix was prepared by solvent stripping. This matrix was used to assess and validate method parameters including linearity, sensitivity, recovery, accuracy and imprecision. Results: HPLC-MS using a binary gradient of methanol/water over 30 minutes achieved resolution of 13 oxysterol compounds and free cholesterol. Initial estimates showed the limit of detection to be approx 4-6 ng/mL with an imprecision of% for hydroxyl oxysterols and ¡Ü20 % for other oxysterols. Stability studies indicated no breakdown or formation of oxysterols during sample preparation steps for 5 of 13 compounds. A blank matrix material consisting of lipid stripped human plasma has been developed. Conclusion: The described method is validated for five oxysterol compounds and is suitable for studies investigating the plasma levels of oxysterols in human health and disease.