Numerous diseases are linked to mutations in the actin-binding domains (ABDs) of conserved cytoskeletal proteins, including β-III-spectrin, α-actinin, filamin, and dystrophin. A β-III-spectrin ABD mutation (L253P) linked to spinocerebellar ataxia type 5 (SCA5) causes a dramatic increase in actin binding. Reducing actin binding of L253P is thus a potential therapeutic approach for SCA5 pathogenesis. Here, we validate a high-throughput screening (HTS) assay to discover potential disrupters of the interaction between the mutant β-III-spectrin ABD and actin in live cells. This assay monitors FRET between fluorescent proteins fused to the mutant ABD and the actin-binding peptide Lifeact, in HEK293-6E cells. Using a specific and high-affinity actin-binding tool compound, swinholide A, we demonstrate HTS compatibility with an excellent Z'-factor of 0.67 ± 0.03. Screening a library of 1280 pharmacologically active compounds in 1536-well plates to determine assay robustness, we demonstrate high reproducibility across plates and across days. We identified nine Hits that reduced FRET between Lifeact and ABD. Four of those Hits were found to reduce Lifeact cosedimentation with actin, thus establishing the potential of our assay for detection of actin-binding modulators. Concurrent to our primary FRET assay, we also developed a high-throughput compatible counter screen to remove undesirable FRET Hits. Using the FRET Hits, we show that our counter screen is sensitive to undesirable compounds that cause cell toxicity or ABD aggregation. Overall, our FRET-based HTS platform sets the stage to screen large compound libraries for modulators of β-III-spectrin, or disease-linked spectrin-related proteins, for therapeutic development.