Photoaffinity labeling is a powerful tool to identify protein targets of biologically active small molecules, yet is often limited,by the size, chemical properties, and availability of photoreactive groups. We report an improved synthesis of photo-leucine, a diazirine-based photoreactive analogue of leucine, and demonstrate its incorporation into a cyclodepsipepticle inhibitor of cotranslational translocation. Photoaffinity labeling in a crude membrane fraction, followed by "click chemistry" with a rhodamine-azide reporter, enabled the identification of Sec61 alpha, the structural core of the Sec61 translocation channel, as the inhibitor's target.