A column-switching high-performance liquid chromatographic method has been developed for the simple and sensitive analysis of BO-2727 (I) in human plasma and urine. Plasma samples were diluted with an equal volume of a stabilizer, and the mixture was directly injected onto the HPLC system. The analyte was enriched in a pre-treatment column, while endogenous components were eluted to waste. The analyte was then backflushed onto an analytical column and quantified with ultraviolet detection. Urinary concentrations were determined in a similar way except that the enriched analyte was eluted in the foreflush mode to a cation-exchange column used for chromatographic separation. The standard curves for the drug were linear in the range of 0.05-50 micrograms/ml in plasma and 0.5-100 micrograms/ml in urine. The limits of quantification for plasma and urine were found to be 0.5 micrograms/ml and 0.5 micrograms/ml, respectively. This method was used to support Phase I clinical pharmacokinetic studies.