To evaluate the optimal treatment scheme and minimize unnecessary use of the highly expensive orphan drug eculizumab, therapeutic drug monitoring should be performed. The aim of this study is to establish a validated ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method to quantify the concentration of eculizumab in human plasma. Plasma samples were prepared using protein G beads, followed by trypsin digestion. A signature peptide for eculizumab (LLIYGATNLADGVPSR) was selected and quantified by UHPLC-MS/MS in the multiple reaction-monitoring mode. The stable isotope-labeled signature peptide *LLIYGATNLADGVPSR (13C6, 15N4-labeled arginine) was used as the internal standard (IS). The UHPLC-MS/MS exhibited good specificity and no matrix effects when employing IS. The calibration curves of eculizumab presented a strong linear relationship from 5 to 1000 µg·mL-1, surpassing the scope of previously published methods and encompassing the concentration levels typically observed in clinical samples. A total of 15 Chinese PNH patients treated with eculizumab were enrolled. The successful application of the method to clinical samples and the data agreement with the ELISA data both demonstrated that established UHPLC-MS/MS is a reliable approach for quantifying eculizumab. Furthermore, this is the first published method used to determine the eculizumab concentration in Chinese PNH patients. The study also suggests that there are large interindividual variations in eculizumab pharmacokinetics among Chinese PNH patients.