Recently, monoclonal antibody (mAb) therapy has gained increasing attention in the medical field due to its high specificity. Dried blood spots (DBSs) have been used in various clinical fields due to their unique characteristics, such as easy transportation, low invasiveness, and home sampling. However, hematocrit (HCT)-associated issues may lead to inaccurate quantification; moreover, the HCT value is required for converting the drug concentration from DBS to plasma. To simultaneously measure HCT levels and quantify mAb concentrations in DBS samples, this study used volumetrically applied 15 μL DBS, and combined protein G purification and ethanol precipitation approaches as the sample preparation method. Sixty-two clinical samples were used to investigate the HCT estimation ability by using hemoglobin (Hb) peptides. Four mAbs, bevacizumab, trastuzumab, nivolumab and tocilizumab, were selected to demonstrate our method, and pembrolizumab was used as the internal standard. The optimized method could measure four mAbs and Hb peptides simultaneously within 11 min. Moreover, a correlation study revealed that the correlation coefficient for the Hb peptides and the HCT value was larger than 0.9. The HCT estimation results revealed that for over 90% of the real DBS samples the HCT could be obtained within ±20% estimation error acceptance criteria. The method was validated in terms of accuracy and precision for the four mAbs. The developed method was further applied to simultaneously quantify mAb concentrations and estimate HCT values in six patient DBS samples to demonstrate its clinical applicability. It is believed that this newly developed method could facilitate various clinical studies and provide benefits for mAb therapies in clinical fields.