Alkyl diazirines are frequently used in photoaffinity labeling to map small molecule-protein interactions in target identification studies. However, the alkyl diazirines can preferentially label acidic amino acids and acidic protein surfaces in a pH-dependent manner, presumably via a reactive alkyl diazo intermediate. Here, we explore the use of ring strain to alter these reactivity preferences and report the development of a cyclobutane diazirine photoaffinity tag with reduced pH-dependent reactivity, termed PALBOX. We show that PALBOX possesses differential reactivity profiles as compared to other diazirine tags in vitro and is readily incorporated into small molecules to profile their binding interactions in cells. Using a set of small molecule fragments and ligands, we show that photoaffinity probes equipped with PALBOX can label the known protein targets in cells with reduced labeling of known alkyl diazirine off-targets. Finally, we demonstrate that ligands equipped with PALBOX can accurately map small molecule-protein binding sites. Thus, PALBOX is a versatile diazirine-based photoaffinity tag for use in the development of chemical probes for photoaffinity labeling experiments, including the study of small molecule-protein interactions.