Kinase-targeted therapy is a new and promising approach to disease treatment. However, some kinase inhibitors have been observed to cause an off-target adverse risk for skeletal system by influencing the growth of osteoblasts. It is known that the proliferation and differentiation of osteoblasts are essentially regulated by MAPK signaling pathway, and many off-target events are considered to influence this pathway. Here, the unexpected MAPK-inhibitor interactions in mouse MC3T3-E1 osteoblastic cells were investigated in detail using an integrative protocol. With bioinformatics analysis we successfully profiled a systematic noncognate interaction spectrum for off-target kinase inhibitors against mouse MAPK kinases, from which 13 potential MAPK-inhibitor interactions were identified. the inhibitors Nilotinib, Dasatinib and Bosutinib were suggested as promising candidates; their cytotoxicity on MC3T3-E1 and inhibitory activity against MAPK kinase were tested at cellular and molecular levels, respectively. We also tested two known MAPK inhibitors SP600125 and SB203580 as positive controls. Consequently, the Dasatinib was found to have high off-target risk for unexpectedly targeting osteoblast MAPK signaling pathway.