Transcription factors and their regulators possess "basic amino acid free domains" which modulate transcriptional gene activation. We aimed at optimizing a MALDI mass spectrometry (MS) analytical method for the characterization of such domains after protein enzymatic digestion. A panel of recombinant transcription factors with different basic residue contents was proteolytically digested with the Asp-N endoprotease and resulting peptide mixtures were analyzed by MALDI-MS with alpha-cyano-4-hydroxy-cinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB) as matrix. We found that peptides without lysine, arginine, histidine (Lys/Arg/His free peptides) were efficiently detected in the positive ion mode only when using DHB. These findings proved to be very useful for two different targeted proteomic applications. Indeed, the MALDI-MS/MS identification of the CARM1 proteolytic cleavage site, which happens in a Lys/Arg/His free domain, could only be achieved using the DHB matrix. Moreover, in routine proteomic analyses, the detection efficiency of Lys/Arg/His free C-terminal peptides of two-dimensional gel separated proteins was strongly enhanced when DHB was used instead of CHCA.