Despite expanded research in stem cell biology, little is known about the mechanisms underlying migration, growth, and differentiation of adipose-derived adult mesenchymal stem cells (ASC). The simultaneous measurement of intracellular pathways opens new avenues to gain further insights in these processes. We used the Cytometric Bead Array (CBA) Flex Set technology to simultaneously analyze protein phosphorylation after stimulation of ASC and compared the results with data generated by corresponding Western blots. Signal transduction of ASC was stimulated by epidermal growth factor (EGF) and analyzed by determining phosphorylation of mitogen-activated protein kinases (MAPKs) ERK, p38, and JNK by Western blotting and CBA. After incubation with EGF, all MAPKs were significantly but differentially phosphorylated depending on time and dose. Furthermore, the ERK-response was abolished by EGF-R antagonist AG 1478 and kinase inhibitor PD98059, whereas p38 and JNK were only inhibited by AG1478. The stimulation and inhibition profiles between the two assays were highly comparable and the data were significantly correlated. In the present study we demonstrated that the CBA technology offers a reliable and convenient method for multiplexing of phospho-proteins in the evaluation of signal transduction pathways of adipose-derived mesenchymal stem cells.