A simultaneous residue analysis of pendimethalin, isopropalin, and butralin in tobacco was developed with high-performance liquid chromatography with ultraviolet monitoring and electrospray ionization mass spectrometry (HPLC-UV-ESI/MS). The herbicide residues of pendimethalin, isopropalin, and butralin in tobaccos were extracted by ultrasonication with ethyl acetate, followed by a cleanup procedure with gel permeation chromatography. The three herbicides were separated within 15 min using a LiChrosorb 18 column with methanol-10 mmol/L ammonium acetate (85:15, v/v) as the eluent. The limits of quantitation, using HPLC-UV, were ca. 0.05, 0.08, and 0.06 mg/kg for pendimethalin, isopropalin, and butralin, respectively, whereas the overall recoveries ranged from 77.5 to 91.8%. The proposed method has been successfully applied to measure 300 real samples, and the residue profiles of three herbicides in tobacco samples were obtained and evaluated.