By comparing the affinities of silica gel, graphitized carbon, C₁₈, and polymer cartridges for the analyzed pesticides, a method for simultaneously determining five neonicotinoid pesticides, including nitenpyram, thiamethoxam, imidacloprid, acetamiprid and thiacloprid, left in tobacco was developed. The sample was extracted with 20% methanol/water under ultrasonic, solid phase extracted through Pesticarb graphitized carbon cartridge and detected by LC/MS/MS. The average recoveries of the pesticides ranged from 80% to 92% with RSD of 2.50% to 4.08% and limit of detection of 0.005 to 0.009 μg/mL. The method is suitable for rapidly determining the five neonicotinoid pesticide residues in tobacco.