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Abstract Regulator of G protein signaling (RGS) proteins play a pivotal role in regulation of G protein‐coupled receptor (GPCR) signaling and are therefore becoming an increasingly important therapeutic target. Recently discovered thiadiazolidinone (TDZD) compounds that target cysteine residues have shown different levels of specificities and potencies for the RGS4 protein, thereby suggesting intrinsic differences in dynamics of this protein upon binding of these compounds. In this work, we investigated using atomistic molecular dynamics (MD) simulations the effect of binding of several small‐molecule inhibitors on perturbations and dynamical motions in RGS4. Specifically, we studied two conformational models of RGS4 in which a buried cysteine residue is solvent‐exposed due to side‐chain motions or due to flexibility in neighboring helices. We found that TDZD compounds with aromatic functional groups perturb the RGS4 structure more than compounds with aliphatic functional groups. Moreover, small‐molecules with aromatic functional groups but lacking sulfur atoms only transiently reside within the protein and spontaneously dissociate to the solvent. We further measured inhibitory effects of TDZD compounds using a protein–protein interaction assay on a single‐cysteine RGS4 protein showing trends in potencies of compounds consistent with our simulation studies. Thermodynamic analyses of RGS4 conformations in the apo‐state and on binding to TDZD compounds revealed links between both conformational models of RGS4. The exposure of cysteine side‐chains appears to facilitate initial binding of TDZD compounds followed by migration of the compound into a bundle of four helices, thereby causing allosteric perturbations in the RGS/Gα protein–protein interface.