Electronic cigarettes (e-cigarettes) are a new form of nicotine delivery device and their popularity is increasing rapidly. One key concern about the safety of e-cigarette use is the quality control during their manufacturing and whether the nicotine content in e-cigarette matches the label claim. We developed an assay to evaluate nicotine content in e-cigarettes. For nicotine extraction, the e-cigarette cartridge consisting of fibrous pad was removed from the cartridge chamber. Ten microlitres of deuterated nicotine (nicotine-d4; 1 μg) solution, used as external standard, was spiked onto the fibrous pad and allowed to penetrate into the matrix. The pad was placed in an Erlenmeyer flask with 50 mL of 50% (v/v) methanol in deionised water as extraction solvent. The flask was placed in an ultra-sound bath for 15 min at ambient temperature. After sonication, the extract was further diluted in Milli-Q water and then analysed using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The recovery of nicotine-d4 extracted from the e-cigarette cartridge was 81.5% ± 3.7 (mean ± SD). Taking into account recovery, nicotine amount in the e-cigarette cartridge samples was 11.9 ± 1.3 mg (mean ± SD), which was lower than the content claimed by the manufacture (16 mg). The nicotine amount did not vary significantly between cartridges within one pack or between packs. The content of nicotine in the e-cigarette cartridges can be effectively determined by UPLC-MS/MS assay. Differences between content and the label claimed for nicotine in e-cigarette could be a potential determinant of clinical trials.