The p38α mitogen-activated protein kinase (MAPK) is essential in controlling the production of many proinflammtory cytokines, and its specific inhibitor can effectively block their production for treating human diseases. To effectively identify highly specific p38α inhibitors in vivo, we developed an ex vivo mouse blood cell-based assay by flow cytometry to measure the intracellular p38α kinase activation. We first attempted to identify the individual blood cell population in which the p38α kinase pathway is highly expressed and activated. Based on CD11b, combined with Ly-6G cell surface expression, we identified two distinct subsets of non-neutrophilic myeloid cells, CD11bMedLy-6G− and CD11bLoLy-6G−, and characterized them as monocytes and natural killer (NK) cells, respectively. Then, we demonstrated that only monocytes, not NK cells, expressed a high level of p38α kinase, which was rapidly activated by anisomycin stimulation as evidenced by the phosphorylation of both p38 and its substrate, MAPKAP-K2 (MK2). Finally, the p38α kinase pathway activation in monocytes was fully inhibited by a highly selective p38α kinase inhibitor dose-dependently in vitro and in vivo. In conclusion, we demonstrated an effective method for separating blood monocytes from other cells and for detecting the expression level and activation of the p38α kinase pathway in monocytes, which provided a new approach for the rapid identification of specific p38α inhibitors.