A procedure for determining trimethoprim in serum is reported. It employs the same high-performance liquid chromatographic system described previously for measurement of anticonvulsants, barbiturates, theophylline, caffeine, acetaminophen, and chloramphenicol in serum. The sample preparation for trimethoprim is nearly identical. As a method extension, quantitation of serum trimethoprim on clinical specimens can be easily accommodated as required.