BACKGROUND To assess the suitability of acetone as an alternative to acetonitrile in SPE under otherwise commonly used conditions, with a focus on selectivity with regard to the most abundant phospholipid class. Two representative analytes were included, leuprolide and tramadol, a peptide and a small molecule, respectively. RESULTS The use of acetone resulted in analogous elution profiles of all monitored compounds in the majority of conditions. The only significant difference was on silica-based C18, where acetone effected markedly enhanced elution of lysophosphatidylcholines than did acetonitrile. Unmodified silica was shown to operate in per aqueous LC mode in highly aqueous conditions. CONCLUSIONS Acetone was established as a selectively similar and slightly more eluotropic alternative to acetonitrile in bioanalytical SPE.