MALDI mass spectrometry imaging (MSI) enables analysis of peptides along with histology. However, there are several critical steps in MALDI MSI of peptides, one of which is spectral quality. Suppression of MALDI matrix clusters by the aid of ammonium salts in MALDI experiments is well-known. It is asserted that addition of ammonium salts dissociates potential matrix adducts and thereafter decreases matrix cluster formation. Consequently, MALDI MS sensitivity and mass accuracy increases. Up to our knowledge, a limited number of MALDI MSI studies used ammonium salts as matrix additives to suppress matrix clusters and enhance peptide signals. In this work, we investigated the effect of ammonium phosphate monobasic (AmP) as alpha-cyano-4-hydroxycinnamic acid (alpha-CHCA) matrix additive in MALDI MSI of peptides. Prior to MALDI MSI, the effect of varying concentrations of AmP in alpha-CHCA were assessed in bovine serum albumin (BSA) tryptic digests and compared with the control (alpha-CHCA without AmP). Based on our data, the addition of AmP as matrix additive decreased matrix cluster formation regardless of its concentration and, specifically 8 mM AmP and 10 mM AmP increased BSA peptide signal intensities. In MALDI MSI of peptides, both 8 mM, and 10 mM AmP in alpha-CHCA improved peptide signals especially in the mass range of m/z 2000 to 3000. In particular, 9 peptide signals were found to have differential intensities within the tissues deposited with AmP in alpha-CHCA (AUC>0.60). To the best of our knowledge, this is the first MALDI MSI of peptides work investigating different concentrations of AmP as alpha-CHCA matrix additive in order to enhance peptide signals in formalin fixed paraffin embedded (FFPE) tissues. Further, AmP as part of alpha-CHCA matrix could enhance protein identifications and support MALDI MSI based proteomic approaches.