Oxysterols in biological samples of limited size are present in low concentrations thus sensitive methods are advantageous. Derivatization of oxysterols prior to liquid chromatography (LC) mass spectrometry (MS) analysis is a common, however a laborious approach. The possibility to reduce the extent of sample preparation by formation of adducts between oxysterols and mobile phase additives (e.g. ammonium acetate) were investigated. Stable adduct ion signals were not obtained with the use of the MS instruments available. However, loss of water ions ([Oxysterol+H-H2O]+ and [Oxysterol+H-2H2O]+, m/z 385.35 and m/z 367.34) were observed when diluting oxysterol standards with ammonium formate (2.5 mM) and formic acid (0.25%) in methanol (MeOH). Under these conditions no easy recognizable fragmentation pattern was observed in tandem MS mode due to clustering of ions in the low mass area when fragmentation energy was applied. Another challenge with the analysis of native oxysterols with a nanoLC system was adsorption to surfaces, especially to fused silica capillaries. Silanization of the fused silica capillaries reduced the issue, but nowhere near satisfactory. It was considered unfeasible to determine native oxysterols with high sensitivity using nanoLC due to the large carry-over issues. Derivatization is therefore recommended when analyzing oxysterols with nanoLC. An on-line automatic filtration filter back-flush solid phase extraction liquid chromatography tandem mass spectrometry (AFFL-SPE-LC-MS/MS) method for determination of Girard T derivatized 24S-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, and 22S-hydroxycholesterol was modified for analysis of exosome samples. Best separation of oxysterol isomers was obtained with an ACE 3 C18 (0.1 mm ID × 150 mm, 3μm, 100 Å) column with a column temperature of 15°C and by using a mobile phase gradient from 0.1/25/75 (v/v/v %) FA/H2O/MeOH to 0.1/10/90 (v/v/v %) FA/H2O/MeOH in 25 minutes. The method was used for analyses of exosome samples obtained with the use of different isolation techniques to find a suitable procedure for exosome isolation.