A major regulatory influence over cell biology is lysine methylation and demethylation within histone proteins. The KDM5/JARID1 sub-family are 2-oxoglutarate and Fe(II)-dependent lysine-specific histone demethylases that are characterized by their Jumonji catalytic domains. This enzyme family is known to facilitate the removal of tri-/di-methyl modifications from lysine 4 of histone H3 (i.e., H3-K4me3/2), a mark associated with active gene expression. As a result, studies to date have revolved around KDM5's influence on disease through their ability to regulate H3-K4me2/3. Recently, evidence has demonstrated that KDM5's may influence disease beyond H3-K4 demethylation, making it critical to further investigate KDM5 demethylation of non-histone proteins. In efforts to help identify potential non-histone substrates for the KDM5 family, we developed a library of 180 permutated peptide substrates (PPS), with sequences that are systematically altered from the WT H3-K4me3 sequence. From this library, we characterized recombinant KDM5A/B/C/D substrate preference. Subsequently we developed recognition motifs for each KDM5 demethylase and used them to predict potential substrates for KDM5A/B/C/D. Demethylation activity was then profiled to generate a list of high/medium/low-ranking substrates for further in vitro validation for each of KDM5A/B/C/D. Through this approach, we analyzed prediction success rate and identified 66 high-ranking substrates in which KDM5 demethylases displayed significant in vitro activity towards. We further shown the ability to monitor changes in cellular methylation in a handful of the 66 high ranking candidate substrates in response to KDM5 inhibition. Specifically, we focused validation efforts on a high-ranking KDM5A novel substrate: p53-K370me3. We demonstrated significant recombinant KDM5A(1-588ΔAP) and KDM5A(1-801) activity towards the p53-K370me3 substrate in vitro. We then monitored KDM5A-mediated demethylation of the p53-K370me3/2 substrate in HCT 116 cells using a combination of wild-type KDM5A and inactive-mutant KDM5A(H483A) overexpression plasmids, along with immunoblotting, (co-) immunoprecipitation and mass spectrometry analysis. Furthermore, we have shown that KDM5A expression influences the established p53-53BP1 interaction. Finally, we identified a novel p53-TAF5 interaction dominated through the p53-K370me3 state and how KDM5A expression might affect this interaction. Ultimately, we have provided the first evidence of a KDM5 demethylase targeting a non-histone substrate for demethylation, via the novel KDM5A demethylation of the p53-K370me3 substrate.