Comparison of a reflective photometry assay (Ames Seralyzer) and a fluorescence polarization immunoassay (Abbott TDx) for measuring phenobarbital and phenytoin serum concentration was performed. Routine phenobarbital and phenytoin plasma levels drawn from patients in the pediatric neurology clinic and pediatric intensive care unit were determined in duplicate by the Abbott TDx and Ames Seralyzer systems. A total of 40 samples were assayed. The interday and intraday variability of the Ames system was determined using calibrators of known concentrations (5–25 mUg/ml). There was significant correlation between the serum phenobarbital or phenytoin concentrations when determined by the Seralyzer and TDx systems. The intraday variability for the measurement of phenytoin when determined by the Seralyzer had coefficients of variation ranging from 2.2 to 8.9%. The interday variability for phenytoin when measured by both the TDx and the Seralyzer correlated well with known calibrators. The utility of the Ames Seralyzer for acute-care facilities, physician offices, and pharmacy satellites is apparent. Based on statistical analysis, the Seralyzer provides accurate phenobarbital and phenytoin serum measurements for clinical use in therapeutic drug monitoring.