Photothermal therapy is achieving ever-increasing attention as a promising method for killing cancer cells. Although, gold nanoparticles are regarded as one of the most effective photothermal therapy agents, the mechanisms underlying their action have to be addressed. Moreover, studies have showed that gold nanoparticles induce apoptosis in treated cultures. Hence, in this study, we investigated the interaction of folic acid functionalized gold nanoparticles and gold-shelled FeO nanoparticles with microtubule and microtubule associated protein tau in order to introduce intracellular targets of these nanoparticles and provide a holistic view about the mechanism of action of gold nanoparticles used in photothermal therapy. Various spectroscopic methods were used to find gold nanoparticles interaction with Tubulin and Tau. Our results indicated that these gold nanoparticles interact with both Tau and Tubulin and their affinity increases as temperature rises. Also, the results illustrated that quenching mechanism for gold nanoparticles interaction with Tubulin and Tau was static. The hydrophobic interaction was determined as driving force for gold nanoparticles binding to Tubulin and Tau. Moreover, it was showed that both type of gold nanoparticles stabilize microtubule polymers. These results suggest Tau and Tubulin as intracellular target of gold nanoparticles and propose that microtubule network is at the heart of apoptosis mechanisms initiated by photothermal therapy.