Protein lysine crotonylation has emerged as an important post-translational modification (PTM) in the regulation of gene transcription through epigenetic mechanisms. Here we introduce a chemical probe, based on a water-soluble phosphine warhead, which reacts with the crotonyl modification. We show that this reagent is complementary to antibody-based tools allowing detection of endogenous cellular proteins such as histones carrying the crotonylation PTM. The tool is also used to show that the histone acylation activity of the transcriptional coactivator, p300, can be activated by pre-existing lysine crotonylation through a positive feedback mechanism. This reagent provides a versatile and sensitive probe for the analysis of this PTM.