The integration of fluorescent microscopy imaging technologies and image analysis into high-content screening (HCS) has been applied throughout the drug discovery pipeline to identify, evaluate, and advance compounds from early lead generation through preclinical candidate selection. In this chapter we describe the development, validation, and implementation of an HCS assay to screen compounds from a kinase-focused small-molecule library to identify inhibitors of the p38 pathway using the GE InCell 3000 automated imaging platform. The assay utilized a genetically modified HeLa cell line stably expressing mitogen-activated, protein-activating protein kinase-2 fused to enhanced green fluorescent protein (MK2–EGFP) and measured the subcellular distribution of the MK2–EGFP as a direct readout of p38 activation. The MK2–EGFP translocation assay performed in 384-well glass bottom microtiter plates exhibited a robust Z-factor of 0.46 and reproducible EC50 and IC50 determinations for activators and inhibitors, respectively. A total of 32,891 compounds were screened in singlicate at 50 μM and 156 were confirmed as inhibitors of p38-mediated MK2–EGFP translocation in follow-up IC50 concentration response curves. Thirty-one compounds exhibited IC50 s less than 1 μM, and at least one novel structural class of p38 inhibitor was identified using this HCA/HCS chemical biology screening approach.