Under certain circumstances, it is necessary to measure both fosphenytoin and phenytoin concentrations. We describe equations by which fosphenytoin concentrations can be calculated accurately by using phenytoin immunoassays. We supplemented aliquots of drug-free serum with fosphenytoin and measured the phenytoin equivalent concentrations (reading a) using fluorescence polarization immunoassay and a chemiluminescent assay. Then 10 microL of alkaline phosphatase (ALP) solution was added to the specimen, and after incubation for 5 minutes at room temperature, total phenytoin concentration was measured (reading b). ALP completely converts fosphenytoin to phenytoin in 5 minutes. Therefore, the delta reading (b-a) represents increased observed value due to complete conversion of fosphenytoin to phenytoin for a particular fosphenytoin concentration. By using the x-axis as the delta reading and the y-axis as the target fosphenytoin concentrations, we observed equations that can be used to calculate the concentration of fosphenytoin in the presence of phenytoin. To test the validity of our equations, we prepared 2 serum pools from patients receiving phenytoin and supplemented them with known concentrations of fosphenytoin. Then initial (reading a) and final (after addition of ALP and incubation, reading b) concentrations were measured by immunoassay. We can accurately predict fosphenytoin and phenytoin concentrations from the delta reading.