This paper describes the direct injection analysis of the anti-cancer drug mitomycin C in biological fluid by an HPLC column switching technique. The first chromatographic column which provides for sample extraction and clean up employs a micellar mobile phase with SDS as the modifier. The second column, coupled on-line to the first, utilizes reverse-phase conditions for analysis. UV detection is employed at 365nm. Samples from cancer patients and dogs were analyzed. The drug recoveries are 95.9–100.3% with a relative standard deviation of 2.76%. A sample analysis is completed within 15 minutes.