Establishing reliable bioanalytical methods is essential to support pharmacokinetic (PK) studies in the preclinical and clinical evaluation of monoclonal antibody (mAb) drugs. Ligand binding assay (LBA) has always been the gold standard for protein quantification, whereas LC-MS has gradually become a promising alternative method for the study of pharmacokinetics of biotherapeutics with its advantages of accuracy and rapid method development. Here, we described for the first time two liquid chromatography-mass spectrometry (LC-MS) methods with different purification pretreatments, protein precipitation and immune affinity (IA) enrichment, along with one electrochemiluminescence (ECL) method for the quantification of an anti-CD47 monoclonal antibody (SHR-1603) in rat and cynomolgus monkey serum. An anti-adsorption reagent was added and digestion conditions were optimized to resolve the absorption issue of hydrophobic peptide in this study. These methods were all validated according to China Food and Drug Administration (CFDA) and European Medicines Agency (EMA) guidelines and were successfully applied to a preclinical study for the quantification of SHR-1603. The respective quantitative ranges of the three methods are respectively 250-500,000 ng/mL (protein precipitation), 100-100,000 ng/mL (IA) and 19.5-10,000 ng/mL (ECL). The two LC-MS methods were compared with ECL method respectively by the cross-validation using the Passing-Bablok regression and Bland-Altman plots. Systematic differences and proportional bias were observed between two LC-MS methods on the one hand and with the ECL method on the other hand. The drug concentrations obtained by the three methods showed good agreement in the low-dose group (ratios of drug exposure, 1.05-1.11), whereas the drug concentrations measured using the LC-MS methods were higher than those obtained by the ECL method in medium-dose and high-dose groups, which can be attributed to the forms of antibodies being determined (free and total). In conclusion, the established LC-MS methods exhibited superior accuracy, efficiency and cost-effectiveness for the PK assessment of SHR-1603 in the preclinical study. Thus, it provides a promising alternative to LBA in pre-clinical and clinical evaluation studies of mAb drugs in various matrices to facilitate the development of anti-tumor drugs.