A sensitive assay of both tegafur (I) and 5-fluorouracil (5-FU) using GLC with a nitrogen-phosphorus-sensitive detector is described. The drugs were extracted from rabbit serum with ethyl acetate and methylated with diazomethane. Linearity was obtained over the concentration ranges of 3.13-200 micrograms/ml for I and 0.0313-2 micrograms/ml and 10-50 ng/ml for 5-FU. The detection limits of I and 5-FU in serum were 50 and 8 ng/ml, respectively. The serum concentrations of the drugs determined by the present method closely agreed with those obtained by spectrophotometry for I and microbial assay for 5-FU.