Phosphoinositides (PIs) play a fundamental role as signalling molecules in various cellular processes. PtdIns(3,4,5)P₃, in particular, is a starting point of important signalling cascades such as PKB/Akt, PKC and PLCγ pathways. Increased PtdIns(3,4,5)P₃ levels are observed in various pathophysiological conditions such as cancer, chronic inflammation and allergy. Inhibition of PI3K activity is thus a promising target for pharmacotherapy. Direct analysis of PIs is traditionally accomplished by labelling with ³H myo-inositol or ³²P_i, and subsequent chromatographic separation and scintillation. These assays are time consuming and expensive, and thus not suited for the fast analysis of large sample numbers. To overcome these limitations we established a rapid LC-MS assay for the separation and quantitative analysis of PtdIns(3,4,5)P₃. The separation of PtdIns(3,4,5)P₃, PtdInsP₂, PtdInsP and PtdIns was achieved in approx. 3 min by ion pair chromatography on a short (2mm x 75 mm) RP-column. As the lipid moieties play no essential role for the biological functions of PIs, analysis of deacylated PIs was preferred. Cells were extracted with chloroform/ methanol/ aqueous HCl according to the standard procedure, followed by deacylation of the phospholipids and LC-MS analysis. HEK 239 cells and neutrophils from whole blood were used to study the changes in PtdIns(3,4,5)P₃ levels obtained upon treatment with different plant substances and extracts. Due to the speed of the assay, it can be integrated into a screening for modulators of PI3K activity.