An online 2-D strong cation exchange (SCX)-RP capillary liquid chromatographic (cLC) method with IT mass spectrometric (IT-MS/MS) detection for the simultaneous determination of prostaglandin (PG) A(1), PGD(2), PGE(1), PGE(2), PGF(2a), 6-keto-(6k)PGF(1a), and 15-Delta(12,14)-deoxy-PGJ(2) (15dPGJ(2)) in cell culture supernatants was developed and validated. Pretreatment of the cell culture supernatants included dilution and filtration, and the analysis time including all sample preparation steps was less than 50 min per sample. Peptides/proteins contained in the matrix were removed by the SCX column. LODs in the range of 0.4-2.2 ng/mL cell culture supernatant, recoveries higher than 80% and within- and between-day precisions of less than 30% RSDs were obtained. Human mesenchymal stem cells (hMSCs) were stimulated with cytokine-containing supernatants derived from activated T lymphocytes, and PG production was analyzed using the developed method. PGE(2 )was found in cultures from both untreated and stimulated hMSCs, while PGE(1) was present above the detection limit only in stimulated cells.