We report the chemical synthesis of the first photo-activatable protein antigen that can be used to study antigen-antibody interaction mediated responses in B cells. This strategy facilitated fine tuning of the caged protein antigen to optimize its bioactivity and photochemical properties. One optimal molecule, HEL-K96NPE, was totally inert to hen egg lysozyme (HEL)-specific B cells and could only restore its antigenicity upon photoactivation. Combined with real time live cell imaging, the utility of HEL-K96NPE was demonstrated as a proof of concept to quantify B cell synapse formation and calcium influx responses at the single cell level.