Phospholipase A 2 (PLA 2 ) quantitation in real-time, using (7-nitro-2-1,3-benzoxadiazol-4-yl)amino-derivatives of phosphatidylcholine (NBD-PCs) as substrates, is influenced by high protein content, color or turbidity. The purpose of the study was to overcome such limitations by a complementary reversed-phase HPLC step to separate the substrates from the products of the reaction. Plasma and bronchoalveolar lavage (BAL) fluid were applied as enzymic sources, while standard porcine PLA 2 and human plasma PAF-acetylhydrolase (PAF-AH) were employed as positive controls. The method was validated with a radiometric assay and compared with the real-time fluorimetric assay. Regarding PLA 2 and PAF-AH determination, the isocratic elution systems CH 3 OH–H 2 O (80:20, v/v) and CH 3 OH–H 2 O–CH 3 COOH (60:40:0.2, v/v/v) separated efficiently C 12 -NBD-FA/C 12 -NBD-PC and C 6 -NBD-FA/C 6 -NBD-PC, with 4.4 and 2.2 resolution, respectively. Analysis time was ∼15min/injection. The reproducibility, expressed as relative standard deviation, was ≤13% for PLA 2 and ≤16% for PAF-AH, respectively. The assay was linear for PLA 2 activities extending from 1pmol up to at least 250nmol FA/h/mL sample, similar to the radiometric assay. It was appropriate for samples with high protein content, where the real-time fluorimetric assay was insufficient. The HPLC method was also evaluated under elevated temperatures, various pH values and Ca 2+ concentrations.