Herein we describe a new enantioselective route for the synthesis of all stereoisomers of 2,3-disubstituted azetidines. This approach uses simple strain release functionalization of preformed 1-azabicyclobutanes (ABBs), which enables installation of a wide array of nucleophiles at the C3-position. Chemical proteomic profiling of stereoisomeric sets of cysteine-reactive acrylamides derived from the new azetidine library reveals ligandable protein sites distinct from those of azetidine probes synthesized using C–H arylation.