Oxysterols are important intermediates in numerous metabolic and catabolic pathways and their biological significance is also proven. The present paper describes a reliable and short liquid chromatography-high-resolution mass spectrometry method (LC-MS/HR-MS) for the quantification of 8 different oxysterols (24(S)-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, 4β-hydroxycholesterol, 7α-hydroxycholesterol, 7β-hydroxycholesterol, 7-ketocholesterol and cholestan-3β,5α,6β-triol) in human plasma and red blood cells. Oxysterols were extracted with iso-octane after saponification of esterified sterols. Due to the poor ionization efficiency of the target compounds in electrospray ionization (ESI) derivatization of the molecules has been performed with N,N-dimethylglycine (DMG). Within less than 8 min we were able to achieve baseline separation of the isobaric 24(S)-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, 4β-hydroxycholesterol, 7α-hydroxycholesterol and 7β-hydroxycholesterol. Moreover, high mass resolution was advantageously applied to resolve quasi-isobaric interferences. The method was validated based on the recommendations of US Food and Drug Administration and the European Medicines Agency guidelines. Oxysterol concentrations were determined in human plasma and red blood cells from healthy volunteers. Furthermore, the applicability for clinical use has been proven by the analysis of oxysterols as biomarkers in Niemann-Pick type C or cerebrotendinous xanthomatosis patients.