Modified antibodies play essential roles in various fields, such as analysis, diagnosis, and therapy, which require modified antibodies with optimal physical and biological properties. To this aim, the development of methods for site-selective antibody modification is crucial. Herein, we introduce a Fab region–selective antibody modification method using epitope-based affinity labeling. Although affinity labeling exploiting the high affinity between an antibody and its epitope may sound straightforward, it remains as a challenge probably due to target affinity loss caused by modification around the epitope binding site. By thoroughly screening of the modifying agent structure, reaction conditions, and purification methods, we achieved to develop an efficient method for the selective modification of the Fab region of the antibody while maintaining its high affinity for the epitope.