Hypericin, a polycyclic aromatic dianthroquinone, is a natural plant product with antiviral properties. We report here the development of a methodology for the extraction and quantitation of hypericin from plasma and biological fluids and the adaptation of a sensitive and selective method for detection of the compound by high-performance liquid chromatography. The methodology offers a rapid and specific means of monitoring drug blood levels in clinical and pharmacokinetic studies. The chromatographic procedure utilizes the substantial retentive properties of hypericin on reversephase media and detection by the strong visible absorbance maximum at 590 nm. Verification by the fluorescence spectral properties of hypericin in organic media can also be utilized. The assay is linear over a 3 log concentration range and hypericin is consistently recovered from murine, simian, and human plasma. The methodology was applied to assess the pharmacokinetic properties of hypericin in mice receiving a single bolus injection of 350 μg. A distribution half-life of 2.0 h and an elimination half-life of 38.5 h were calculated. We also discuss the limitations of direct analysis of hypericin by absorbance or fluorescence measurements.