Herein, we outline the protocol for application of 4-(2-((4-bromophenethyl)dimethylammonium)ethoxy)benzenaminium dibromide (4-APEBA) which we have previously demonstrated to be an effect reagent in tandem with 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC) to target carbonyl containing metabolites in-situ for matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). Either with or without the selective application of EDC, MALDI-MSI is able to differentiate ketones and aldehydes from carboxylic acids lending more confidence to annotations. This has enabled metabolic pathway analyses with high-levels of coverage routinely enabling the detect of over a hundred metabolites containing carbonyls. This reagent was previous introduced for LC-MS/MS purposes (see references), but generally through our on-tissue/on-target chemical derivatization (OTCD) protocol we find that (1) we have increased sensitivity and coverage of primary metabolites, (2) we have increased confidence within our annotations due to bromine isotopic patterns, (3) we do not delocalize metabolites within the process of OTCD, (4) we have multiplexed detection of non-derivatized metabolites and lipids in tandem with OTCD products, and (5) this is applicable to a broad variety of mammalian or plant tissues, and microbial cultures.