This study describes, for the first time, the development of an automated sensitive flow fluorescent noncompetitive immunoassay based on kinetic-exclusion analysis (KinExA) for the quantitative determination of human plasma levels of monoclonal antibodies (mAbs) used for cancer immunotherapy. The assay was adapted on KinExATM 3200 biosensor and optimized and validated for bevacizumab (BEV) and cetuximab (CET), as representative examples of the mAbs, using their specific antigens. These antigens were the human vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR) for BEV and CET, respectively. The limits of detection were 1.28 and 52.64 ng mL−1 for BEV and CET, respectively. The accuracy of the assay was demonstrated with analytical recovery of analytes from spiked plasma at 96.2 – 104.3 and 96.8 – 105.3% for of BEV and CET, respectively. The precision of the assay was satisfactory as shown by relative standard deviation (RSD) at 2.2 – 5.7 and 2.5 – 6.1% for assay of BEV and CET, respectively. The high sensitivity of the assay allowed the use of very small volumes (~ 1 μL) of plasma sample for analysis. Automated analysis by the proposed KinExA-based assay facilitates the processing of large numbers of mAbs-containing specimens in studies of pharmacokinetics (PK), pharmacodynamics (PD), and therapeutic drug monitoring (TDM) of therapeutic mAbs. The proposed assay can be used to overcome the problems encountered in the existing conventional immunoassays for mAbs.