The complexity of the p38 mitogen-activated protein kinase (MAPK) signaling pathway presents challenges to understanding the efficacy of p38 inhibitors. Biochemical recombinant kinase assays and tumor necrosis factor alpha (TNFalpha) secretion assays are typically used to evaluate p38alpha inhibitors, but they do not provide insight into proximal intracellular events. Stimulation of the pathway evokes a cascade of phosphorylation events, accompanied by movement of molecules to different cellular compartments. Herein, we describe the profiling and potency comparison of a large set of p38alpha inhibitors with a pyrimidinone, imidazopyrimidine, or triazolopyrimidine core against biochemical recombinant p38alpha kinase activity, lipopolysaccharide (LPS)-mediated TNFalpha secretion by THP-1 cells, and a set of cellular imaging assays in SW1353 chondrocytes and baby hamster kidney cells. These pathway assays included p38 phosphorylation, MAPK-activated protein kinase 2 translocation, and heat shock protein (HSP) 27 phosphorylation. We established that HSP27 phosphorylation correlates well with LPS-induced TNFalpha secretion, validating our cellular imaging assays. We also found that the choice of cells and inducer can profoundly affect cellular potency results. High-content analysis may reveal signaling details, enriching our understanding of the mechanism of action of p38alpha inhibitors.