A new high-performance liquid chromatographic method with column switching has been developed for the determination of omeprazole in plasma. The plasma samples were injected onto a Bondapak phenyl/corasil (37-50 microns) precolumn and polar plasma components were washed with 0.06 M borate buffer. After valve switching, the concentrated drug were eluted in the back-flush mode and separated on a mu-Bondapak C18 column with acetonitrile-phosphate buffer as the mobile phase. The method showed excellent precision, accuracy and speed with detection limit of 0.01 microgram/ml-1. Total analysis time per sample was less than 20 min and the coefficients of variation for intra and inter-assay were less than 5.63%. This method has been successfully applied to plasma samples from rats after oral administration of omeprazole.