Acetyl-CoA:lysoplatelet-activating factor (1-O-alkyl-sn-glycero-3-phosphocholine) acetyltransferase (lysoPAF-AT) (EC 2.3.1.67) is a key enzyme in the biosynthesis of platelet-activating factor (PAF) and has been shown to be activated by various extracellular stimuli. A novel method to determine the enzyme activity is described here, which enables 96 simultaneous assays in a standard 96-well microplate format. The assay is based on the quantification of the incorporation of [³H]acetyl-CoA into PAF in the presence of lysoPAF. The radioactive products are separated from the substrate with a 96-well-formatted chromatography device using a Multiscreen plate (Millipore) prefilled with octyl-silica gel. As little as 1 mg octyl-silica gel was sufficient for the efficient recovery of the radioactive product, resulting in the very low background and thus high sensitivity. The enzyme activity could be measured directly with whole cell lysates from various cells cultured in 96-well microplate scale. This tailor-made microplate chromatography separation step is readily applicable for other kinds of enzyme assays.