Pyridoxal kinases (PLK) are crucial enzymes for the biosynthesis of pyridoxal phosphate, an important cofactor in a plethora of enzymatic reactions. The evolution of these enzymes resulted in different catalytic designs. In addition to the active site, the relevance of a cysteine, embedded within a distant flexible lid region, was recently shown. This cysteine forms a hemithioacetal with the pyridoxal aldehyde and is essential for catalysis. Despite the prevalence of these enzymes in various organisms, no tools were available to assess the general relevance of the lid residue. We here introduce pyridoxal probes equipped with an electrophilic trapping group in place of the aldehyde which readily reacts with reactive lid cysteines as a mimic of hemithioacetal formation. This irreversible capture not only facilitates the readout of cysteine reactivity but also the enrichment of PLKs from living cells via alkyne handles placed at two different positions within the pyridoxal structure. Interestingly, depending on the position, the probes either displayed a preference for Gram-positive or Gram-negative PLK enrichment in living cells. By applying the cofactor traps, we were able to validate not only previously investigated Staphylococcus aureus and Enterococcus faecalis PLKs but also Escherichia coli and Pseudomonas aeruginosa PLKs unravelling a crucial role of the lid cysteine for catalysis. Overall, our tailored probes facilitated a reliable readout of lid cysteine containing PLKs and thereby qualify them as chemical tools for further mining diverse proteomes for this important enzyme class.