The importance of thiols has stimulated the development of a number of methods for determining glutathione and other biologically significant thiols. Methods that are currently available, however have some limitations, such as being time consuming and complex. In the present study, a new high-performance liquid chromatography (HPLC) method for determining biological thiols was developed by using 9-Acetoxy-2-(4-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)phenyl)-3-oxo-3H-naphtho[2,1-b]pyran (ThioGlo3) as a derivatizing agent. ThioGlo reacts selectively and rapidly with the thiols to yield fluorescent adducts which can be detected fluorimetrically (lambda(ex) = 365 nm, lambda(em) = 445 nm). The within-run coefficient of variation for glutathione (GSH) by this method ranges from 1.08 to 2.94% whereas the between-run coefficient of variation for GSH is 4.31-8.61%. For GSH, the detection limit is around 50 fmol and the GSH derivatives remain stable for 1 month, if kept at 4 degrees C. Results for GSSG and cysteine are also included. The ThioGlo method is compared to our previous method in which N-(1-pyrenyl)maleimide (NPM) is used to derivatize thiol-containing compounds. The present method offers various advantages over the currently accepted techniques, including speed and sensitivity.