A fully enzymatic assay is described for the determination of triglycerides. The coupled activities of triacylglycerol acylhydrolase and glycerol kinase result in the formation of glycerol-3-phosphate. The system also contains L-alpha-glycerol-phosphate oxidase, which produces hydrogen peroxide from glycerol-3-phosphate, and a sensitive chromogenic indicator system, consisting of peroxidase, 4-chlorophenol and 4-aminophenazone. We evaluated this method with respect to kinetics, linearity, blank rates, precision, accuracy, reagent stability and interfering substances. The accuracy of the triglyceride assay demands that each enzymatic reaction step be complete and homogeneous. We therefore developed HPTLC-1) and HPLC-2) methods to monitor the course and completeness of each step.