Pancreatic lipase is produced and stored in pancreatic acinar cells, and is normally secreted into the duct system. In disorders that cause pancreatic damage, pancreatic lipase enters the circulation, and serum lipase activity becomes useful in the diagnosis and evaluation of pancreatitis. During the last decade, many lipase assays have used the Imamura method, in which a series of enzymatic reactions utilizing a diglyceride substrate leads to the formation of a colorimetrically detected product. Historically, this method has been prone to significant interference from various substances, including glycerol itself. In the light of these limitations, we evaluated an automated enzymatic rate assay that uses a non-diglyceride-based, pancreatic lipase-specific substrate. Precision, linearity, and potential interference were assessed, and when compared to the Imamura method, the non-diglyceride-based assay exhibited a slope of 0.475, y-intercept of 15.89, r-value of 0.9516, and S(y,x) of 12.96. Similar results were also observed when the two assays were compared using samples with markedly elevated creatinine levels. Between-day coefficients of variance (CVs) ranged from 5.0% to 5.5%, which compared well with the diglyceride-based method, and linearity spanned a range of 3-156 U/L. Evaluation of over 2,000 patient results collected during a 6-month period suggested that the manufacturer's upper reference limit of 51 U/L may be too conservative. No significant interference was identified with bilirubin, triglyceride, or glycerol itself, and significant interference from hemoglobin was observed only at concentrations of 116 mg/dL or greater. As a result, the non-diglyceride-based method provides an acceptable alternative for the routine laboratory measurement of lipase activity.