Development of modulators of constitutively active, kinase domain mutants of c-Kit has proved to be very difficult. Therefore, a high-throughput differential cytotoxicity assay was developed to screen for compounds that preferentially kill cells expressing constitutively active c-Kit. The cells used in the assay, murine IC2 mast cells, express either the D814Y activating mutation (functionally equivalent to human D816Y) or wild type protein. This assay is robust and highly reproducible. When applied to libraries of natural product extracts (followed by assay-guided fractionation), two differentially active compounds were identified. To assess possible mechanisms of action, the active compounds were tested for inhibitory activity against a panel of signaling enzymes (including wild type and mutant c-Kit). Neither of the compounds significantly affected any of the 73 enzymes tested. The effects of commercially available modulators of known signaling components were also assessed using the screening assay. None of these inhibitors reproduced the differential activity seen with the natural products. Finally, both compounds were found to affect mitochondrial potential in cells expressing c-Kit(D814Y). These results suggest that the newly identified natural products may provide new avenues for intervention in aberrant c-Kit signaling pathways.