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Abstract Proliferating cell nuclear antigen (PCNA) is a homo‐trimeric protein complex that clamps around DNA to tether DNA polymerases to the template during replication and serves as a hub for many other interacting proteins. It regulates DNA metabolic processes and other vital cellar functions through the binding of proteins having short linear motifs (SLiMs) like the PIP‐box (PCNA‐interacting protein‐box) or the APIM (AlkB homolog 2 PCNA‐interacting motif) in the hydrophobic pocket where SLiMs bind. However, overproducing TbPCNA or human PCNA (hPCNA) in the pathogenic protistTrypanosoma bruceitriggers a dominant‐negative phenotype of arrested proliferation. The mechanism for arrestingT. bruceiproliferation requires the overproduced PCNA orthologs to have functional intact SLiM‐binding pocket. Sight‐directed mutagenesis studies showed thatT. bruceioverproducing PCNA variants with disrupted SLiM‐binding pockets grew normally. We hypothesized that chemically disrupting the SLiM‐binding pocket would restore proliferation inT. brucei, overproducing PCNA orthologs. Testing this hypothesis is the proof‐of‐concept for aT. brucei‐based PCNA screening assay. The assay design is to discover bioactive small molecules that restore proliferation inT. bruceistrains that overproduce PCNA orthologs, likely by disrupting interactions in the SLiM‐binding pocket. The pilot screen for this assay discovered two hit compounds that linked to predetermined PCNA targets. Compound#1, a known hPCNA inhibitor, had selective bioactivity to hPCNA overproduced inT. brucei, validating the assay. Compound#6had promiscuous bioactivity for hPCNA and TbPCNA but is the first compound discovered with bioactivity for inhibiting TbPCNA.