.....Preanalytical validation is a crucial point to minimize the susceptibility of oxysterols to in vitro autoxidation. The aim of this study was to standardize a preanalytical protocol for ROS-derived oxysterol analysis by liquid chromatography-tandem mass spectrometry in human plasma. Sample matrices were compared and stability of free oxysterols in whole blood and EDTA-plasma was investigated with regard to short-term storage until sample preparation, freeze-thaw cycles, addition of butylated hydroxytoluene and long-term storage up to 1year at different temperatures (-20°C, -80°C and -130°C) as well as different storage containers (safe-lock tubes, cryo tubes and straws). Sample preparation prior LC-MS/MS analysis was reduced to a simple concentration and protein precipitation step. Storing EDTA-whole blood for 30min at room temperature resulted in <25% concentration changes, within acceptable change limits (ACL). In freshly prepared plasma samples, free oxysterols were stable for 90min stored at 4°C with concentration changes <23.5% (within ACL). Up to nine freeze-thaw cycles did not affect analyte concentrations (concentration change -8.5% to +5.0%). 7-Ketocholesterol was stable for 2years stored <-80°C; concentration changes below 20.5% (within ACL). The remaining oxysterols were stored for a maximum of 2-4weeks without exceeding ACL. The addition of BHT did not reveal improvement in analyte stability for storage at -80 or -130°C. We developed a standardized preanalytical protocol for oxysterol analysis based on LC-MS/MS, compared cryobanking conditions for each oxysterol and present data for long-term storage up to 2years.