The CellSensor® AP1-bla A375 cell line contains a beta-lactamase reporter gene under control of the AP1 response element stably integrated into A375 cells. A375 cells are human melanoma cancer cells that contain the endogenous B-Raf mutation V600E resulting in constitutive B-Raf kinase activity. The CellSensor® AP1-bla A375 cell line is a clonal population isolated by flow cytometry based on constitutive expression of beta-lactamase. This cell line has been validated with various small-molecule Raf inhibitors as well as B-Raf Stealth RNAi™ siRNA. This cell line has also been tested for assay performance under variable conditions, including DMSO concentration, cell number, compound incubation time, and substrate loading time and validated for Zfi-factor and IC50 concentrations of Raf1 Inhibitor I. The RAF gene family (RAF1, A-RAF, and B-RAF) encodes closely related serine/threonine protein kinases that are important effectors of Ras activation. Raf1 and A-Raf are rarely mutated, whereas mutations in B-Raf are common in human cancers, especially melanoma. B-Raf is mutated in about 70% of human melanomas, 35-70% of papillary thyroid carcinomas, and less commonly in lung and colorectal carcinomas. Mutations are mostly in the B-Raf kinase domain and, in melanomas, the vast majority are V600E missense mutations leading to activation of B-Raf kinase. The constitutive activity of B-Raf V600E can directly lead to the activation of the Mek/MAPK signaling pathway. Therefore, inhibition of B-Raf/Mek/MAPK signaling could be a potential way of treating melanomas and other tumors with mutant B-Raf.