A sensitive and selective method for quantitation of erdosteine in human plasma was established by use of 96-well solid-phase extraction (SPE) and liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI/MS/MS). Plasma samples were transferred into 96-well OASIS HLB extraction plate using an automated sample handling system and the drugs were eluted with methanol. The eluents were then evaporated and reconstituted with mobile phase. All sample transfer and SPE was automated through the application of both the Perkin-Elmer MultiPROBE II HT and TOMTEC Quadra 96 workstation. Compounds were separated on a C18 column with 1 mM ammonium acetate-acetonitrile (80:20, pH 3.2), as mobile phase at a flow rate of 0.3 ml/min. The limit of quantitation (LOQ) was 0.2 ng/ml, using a sample volume of 0.2 ml for the analysis. The reproducibility of the method was evaluated by analyzing three at 14 quality control (QC) levels over the nominal concentration range from 0.2 to 5000 ng/ml. The intraday accuracy was found to range from 99.6 to 105.0% with precision (% RSD) of less than 4.76% at five QC levels. The interday accuracy was found to range from 95.0 to 100.5% with precision of less than 5.26% at five QC levels. Erdosteine produced a protonated precursor ion ([M+H]+) at m/z 250, and a corresponding product ion at m/z 204. Internal standard (letosteine) produced a protonated precursor ion ([M+H]+) at m/z 280 and a corresponding product ion at m/z 160. The high sample throughput of the method has been successfully applied to a pharmacokinetic study of erdosteine in human plasma.