A simple, sensitive and rapid LC/MS/MS method was developed for the quantification of lansoprazole in human plasma. After a simple sample preparation procedure by one-step protein precipitation with acetonitrile, lansoprazole and the internal standard bicalutamide were chromatographed on a Zorbax SB-C18 (3.0 mm × 150 mm, 3.5 μm, Agilent) column with the mobile phase consisted of methanol–water (70:30, v/v, containing 5 mM ammonium formate, pH was adjusted to 7.85 by 1% ammonia solution). Detection was performed on a triple quadrupole tandem mass spectrometry by multiple reaction monitoring (MRM) mode via negative eletrospray ionization source (ESI−). The lower limit of quantification was 5.5 ng/mL, and the assay exhibited a linear range of 5.5–2200.0 ng/mL. The validated method was successfully applied to investigate the bioequivalence between two kinds of preparation (test vs. reference product) in twenty-eight healthy male Chinese volunteers.