A rapid method to quantify levels of the beta-thioglycoside N-hydroxyl sulfate, glucoraphanin, in dog and rat plasma to support pre-clinical toxicological and pharmacological studies has been developed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Glucoraphanin was extracted from plasma by protein precipitation with acetonitrile and separated via hydrophilic interaction liquid chromatography (HILIC) using a Luna 5microm Silica (2) 100A column (50mmx2.0mm) at a flow rate of 0.3mL/min. Solvent A consisted of 200mM ammonium acetate and formic acid (99:1, v/v) and Solvent B was acetonitrile. Initial conditions (90% Solvent B) were held for 0.01min after injection, decreased to 40% in 0.5min and held constant for 2.5min, returning to initial conditions for 3min (reequilibration time). Glucoraphanin was detected by MS/MS using a turbo ion spray interface as the ion source operating in negative ion mode. Acquisition was performed in multiple reaction monitoring mode at m/z 435.8-->96.7. The method was validated for the calibration range 10-2000ng/mL. Within- and between-run precision for the low, mid and high QC levels was 8% R.S.D. or less and accuracy ranged from 100 to 113%. The lower limit of quantification was 10ng/mL; calibration curves encompassed the range of plasma concentrations expected to be found in bioavailability and pharmacokinetics studies with glucoraphanin. The method has successfully been applied to the determination of glucoraphanin in dog and rat plasma and should be extendable to other species as well.