A new orally active inotropic agent, 6-[3,4-dihydro-3-methyl-(2H)-2-oxoquinozolinyl)]-4,5-dihydro-3- (2H)-pyridazinone (I), is currently under investigation. In support of clinical studies, a HPLC assay for the analysis of compound I in human plasma has been developed. The method involved a solid-phase extraction using C18 cartridge columns washed with methanol-water (20:80) and eluted with acetonitrile-water (70:30). The eluate was then extracted with dichloromethane. A reversed-phase alkylphenyl-bonded column was used as the analytical column. The mobile phase was a mixture of methanol, acetonitrile, 2-propanol and phosphate buffer (pH 4.6). A wavelength of 311 nm was used for detection. The limit of detection of the assay was 2 ng/ml, and the limit of quantitation was 5 ng/ml. A linear calibration range of 5 ng/ml to 1200 ng/ml was obtained with a correlation coefficient less than 0.99. The precision and accuracy were evaluated by analyzing samples of three different concentrations (n = 5), 40, 200 and 800 ng/ml in plasma. The coefficients of variation and the differences from nominal values were less than 10%. The average recovery for 5, 50 and 100 ng/ml of analyte in plasma was about 90%. This assay has been applied to clinical studies with satisfactory performance and to plasma of different species in preclinical studies.