Phenytoin is a commonly used anticonvulsant that is highly protein bound with a narrow therapeutic range. The unbound fraction, free phenytoin (FP), is responsible for pharmacologic effects; therefore, it is essential to measure both FP and total serum phenytoin levels. Historically, the Abbott TDx method has been widely used for the measurement of FP and was the method used in our laboratory. However, the FP TDx assay was recently discontinued by the manufacturer, so we had to develop an alternative methodology. We evaluated the Beckman-Coulter DxC800 based FP method for linearity, analytical sensitivity, and precision. The analytical measurement range of the method was 0.41 to 5.30 microg/mL. Within-run and between-run precision studies yielded CVs of 3.8% and 5.5%, respectively. The method compared favorably with the TDx method, yielding the following regression equation: DxC800 = 0.9**TDx + 0.10; r2 = 0.97 (n = 97). The new FP assay appears to be an acceptable alternative to the TDx method.