This study addresses a technique to explore mechanical role of intracellular structures by using a triton cytoskeleton model (TC model) for traction force measurements. Inhibition of signaling pathways of myosin light chain phosphorylation, possibly being induced by disruption of mictotubules, is also performed. Traction forces for the TC model significantly decreased compared to control. In contrast, traction forces significantly increased from 10.3 ± 2.5 nN to 13.3 ± 3.7 nN after treatment of nocodazole, which is well consistent with previous studies. From these results, not only cytoskeletal structures but also other cellular components such as cytoplasm should be involved in cell mechanics. Separate fluorescence studies showed that microtubules disruption induced myosin light chain phosphorylation. Exposure to Y27632 showed that traction forces decreased by 80% compared to control within 15 min and the following treatment with nocodazole showed only 40% recovery from the priori decreased forces. This result indicates that microtubules disassembly may modulate the actomyosin mechanism leading to the increase in traction forces, mainly through the ROCK pathway. It can be concluded that contribution of microtubules should include not only a force balance but also a modulator of the actomyosin-based contractile system.