Overexpression of c-MYC protein without genomic amplification occurs in multiple cancers, including neuroblastoma and small cell lung cancer. In searching for the mechanisms of c-MYC protein overexpression, we demonstrated that the transcription factor, OCT4 mediates c-MYC transcriptional activation in progressive disease neuroblastoma. Subsequently, we identified two kinases, MAPKAPK2 (MK2) and DNA-PK, which are predicted to bind and phosphorylate OCT4 at S111 and S93 residues, respectively. Based on these novel observations, we developed a cell-based luminescence assay to screen and identify compounds that inhibit the interactions between MK2 and OCT4. By screening 79,671 compounds, we identified 65 compounds we designated as “hits”. Using a two-step validation of co-immunoprecipitation and pOCT4S¹¹¹ detection, the compounds were further narrowed down to three for further studies. The three compounds were tested for their ability to inhibit kinase activity, in vitro cytotoxic activity, and anti-inflammatory activity. In conclusion, we developed a cell-based luminescence assay for the discovery of new agents targeting the c-MYC transcriptional activation pathway. Screening and subsequent validation identified a small number of compounds for further development.