The extracellular signal-regulated kinase (ERK1 and ERK2) proteins play an important role in the proliferation and survival of many types of cancer cells. However, no specific inhibitors of ERK proteins are currently available. Drug discovery efforts focusing on kinase inhibitors target the ATP binding site and the catalytic domain. However, the conserved features of the ATP binding site among protein kinases make identifying a selective inhibitor difficult. Therefore, it is important to develop ATP-independent inhibitors that block the functions of active ERK2 in cancer cells. Different from traditional methods which screen compounds from large chemical libraries, we used the 3D structure of active ERK2 and computer aided drug design (CADD) to identify novel compounds that selectively prevent ERK-substrate interactions. ATP-independent ERK docking domain inhibitors were initially selected in silico, and then were characterized by in vitro cell based assays. The results indicated that several test compounds bind to ERK2, and inhibit ERK substrate-specific phosphorylation and cancer cell proliferation. Subsequently, real time binding interactions between ERK2 and substrates were measured using surface plasmon resonance (SPR). Effects of an identified ERK2 inhibitor, compound 76, on substrate interactions were evaluated. Lastly, Caenorhabditis elegans (C. elegans) was chosen as a research model in order to examine the potency of ERK2 inhibitors in a whole organism. C. elegans is a well characterized organism that is inexpensive and easy to maintain in the laboratory. More importantly, C. elegans has the MPK-1 protein, which is homologous to human ERK2, known to be required for vulva formation and egg laying. It was shown that preincubation of C. elegans eggs or larvae with several compounds causes inhibition of egg laying. This effect was due to compound 76 inhibition of proper vulva cell fusion during development. In conclusion, CADD and biological assays can be used to identify lead compounds that are selective for active ERK2 and its interactions with substrates involved in cancer cell proliferation and normal physiological functions.