The application of immunocapture LC-MS/MS in drug testing has expanded considerably over the past decade, offering significant advantages in analysis, development, and identification. In this study, we developed and validated a universal immunocapture LC-MS/MS assay for the detection of humanized IgG1-based drugs in cynomolgus monkey sera. The method selected a 12-amino acid characteristic peptide from the CH1 region of IgG1 as a quantification surrogate and used the heavy stable isotope-labeled same peptide as an internal standard. Furthermore, humanized IgG1-based drugs were immunocaptured using magnetic streptavidin beads pre-bound to biotinylated anti-human IgG1 Fc antibodies, followed by trypsin digestion. The related liquid chromatography was conducted on a Shiseido C18 column (2.0 mm × 100 mm, 3 μm) equipped on a Shimadzu Nexera X2 UHPLC system. Quantification of the humanized IgG1 -based drugs was performed on a LCMS-8050 triple quadrupole mass spectrometer using multiple reaction monitoring (MRM) mode with positive electrospray ionization. The developed assay was validated for parameters including calibration curve, precision, accuracy, selectivity, recovery, and stability. The obtained results all met the acceptance criteria valid for ligand-binding assays (LBAs). Besides, involving humanized IgG1 -based drugs exhibited good stability in cynomolgus monkey sera under different tested conditions. After validation, the assay was successfully applied to the pharmacokinetic studies of two humanized IgG1-based antibody-drug conjugates (ADCs), respectively, generating comparable pharmacokinetic profiles to related LBAs. Together, the results confirm the assay's reliability, universality, and feasibility for quantifying the target drugs.