Tanshinone IIA (TS) and cryptotanshinone (CT) are the major active constituents contained in Radix salvia miltiorrhiza. This paper described a rapid, sensitive and specific assay for the simultaneous quantitative determination of TS and CT in rat plasma. After a single step of liquid-liquid extraction, plasma samples were analyzed by high performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) using a reversed-phase C18 column (150 mmx2.0 mm, 5 microm, Shim-pack VP-ODS column). The assay was linear in the concentration range of 2-200 ng/ml. The lower limits of quantification of TS and CT were 1 and 0.2 ng/ml, respectively. Recoveries of TS and CT were greater than 80%. The precisions and accuracies determined from 5 days were all within 12%. The assay was applied to a pharmacokinetic study in rats after an oral administration of total tanshinones with a dose of 150 mg/kg (containing 12% of TS and CT). Results showed that this simple and rapid method was sensitive enough to follow the plasma levels of TS and CT in rats, even though the concentration maximums of both were below 20 ng/ml after an oral administration of total tanshinones.