Protein thiocarboxylates are involved in the biosynthesis of thiamin, molybdopterin, thioquinolobactin, and cysteine. Sequence analysis suggests that this post-translational modification is widely distributed in bacteria. Here we describe the development of lissamine rhodamine B sulfonyl azide as a sensitive click reagent for the detection of protein thiocarboxylates and describe the use of this reagent to detect PdtH, a putative protein thiocarboxylate involved in the biosynthesis of the pyridine dithiocarboxylic acid siderophore, in the Pseudomonas stutzeri proteome.