We have attempted a high throughput-screening to find differentiation modifying compounds of keratinocyte. 11 inhibitors of known signaling pathway and approximately 4000 chemical compounds were screened using an In Cell Western system based on immunofluorescent staining. Staurosporine, a protein kinase C inhibitor and H89, a protein kinase A inhibitor, promote the expression of involucrin, a terminal differentiation marker in keratinocytes. On the other hand, U0126, a MEK1 specific inhibitor, and SAHA or SBHA, two HDAC inhibitors, prevent the expression of involucrin during calcium induced differentiation. In addition, we have found a novel compound that induces differentiation and two compounds that inhibit the differentiation of keratinocytes. One of these compounds also induced differentiation of a SCC cell line as well as apoptosis with a lower IC50 than that towards primary keratinocyte. These compounds might be useful in the future as tools to study differentiation of stratified epithelium as well as putative anti-tumor targets.