Objective To investigate the impact of different cell digestion methods on the binding of a humanized monoclonal antibody targeting CD3L1 （designated as 5H） to tumor cells. Methods Trypsin solution and a collagenase/neutral protease cocktail， combined with two digestion temperatures （room temperature and 37°C）， were used to dissociate， passage， and collect various adherent tumor cell lines. Flow cytometry and confocal microscopy were then employed to compare differences in 5H antibody binding under different cell digestion conditions. Results Cells dissociated and passaged with trypsin solution at room temperature exhibited only weak binding to the 5H antibody， whereas those treated with the collagenase/neutral protease cocktail at room temperature showed significantly stronger binding. There were notable differences in the binding signal and the proportion of antibody-stained positive cells between the two groups （P<0.01）. Additionally， cells treated with the collagenase/neutral protease cocktail exhibited antibody binding signals that were dependent on the concentration of the 5H antibody， enabling receptor occupancy analysis. Compared to digestion at room temperature， treatment at 37°C resulted in a slight reduction in the binding of target cells to the 5H antibody， however， this difference was not statistically significant. Conclusion Trypsin digestion significantly reduces the binding of the anti-CD3L1 antibody to tumor cells， whereas the collagenase/neutral protease cocktail effectively preserves it. With an appropriate choice of digestion enzymes， the impact of digestion temperature is relatively minor. These findings suggest that optimized cell digestion protocols should be employed when studying interactions between antibody drugs and cell surface antigens.