A novel method has been developed for the determination of cefaclor in human plasma by ultra-performance liquid chromatography combined with tandem mass spectrometry (UPLC-MS-MS). The plasma was treated by a single step of protein precipitation with acetonitrile. The chromatographic separation was performed on a Waters Acquity UPLC BEH C18 (2.1 × 100 mm, 1.7 µm) with a gradient mobile phase consisting of 0.1% formic acid and acetonitrile at a flow rate of 0.4 mL/min. The analyses were conducted by multiple reaction monitoring using the precursor-to-product combinations of m/z 367.5 → 173.8 (cefaclor) and m/z 454.1 → 160.3 (internal standard). Validation results indicated that the lower limit of quantification was 2 ng/mL and the assay exhibited a linear range of 2-10,000 ng/mL. Quality control samples (5, 200 and 5,000 ng/mL) in five replicates from three different runs of analysis demonstrated an intra-assay precision (relative standard deviation) of 3.7-10.7%, an inter-assay precision of 5.8-8.9%, and an overall accuracy of < 15%. A sensitive and specific method for quantifying cefaclor in human plasma has been devised and successfully applied to a pharmacokinetic study.