To determine the IC50 of ERK phosphorylation inhibitors, we run a titration on the concentration of the test compound; here we use the drug Sorafenib as an example. The IC50 of Sorafenib, an ERK phosphorylation inhibitor, was determined for three cell lines: PANC-1, NIH-3T3, and RBL-2H3. The three cell lines were plated in sterile, black 96-well plates at a cell density of 10,000 cells per well and allowed to adhere overnight. The culture medium was aspirated and replaced with culture medium dosed with Sorafenib at the following concentrations: 10-2, 10-4, 10-5, 10-6, 10-7, 10-8, and 10-9 M. The dosed culture medium was prepared from a stock solution of Sorafenib in DMSO so that the final concentration of DMSO in the medium was 1%. Negative control wells received Sorafenib free culture medium with 1% DMSO. All treatment conditions were run in triplicate. The cells were incubated for three hours with the Sorafenib. After the three hour incubation, the Sorafenib medium was aspirated and replaced with 100 ng/mL PMA dosed medium. The cells were incubated with the PMA medium for five minutes. The pERK and total protein was then quantified using the ERK Phosphorylation Assay Kit (EERK-100). The pERK values were normalized to the total protein content of the sample.