Nitrotyrosine, a structural analogue of tyrosine, is present in cells in pathological conditions and is incorporated into tubulin to form tubulin tyrosine nitration, which disrupts the normal function of microtubules. There is limited research on the functional aspects of tubulin tyrosine nitration in different types of tumor. In the present study, the effect of tubulin tyrosine nitration and tubulin tyrosine ligase like 12 (TTLL12) on the proliferation of SCC-25 cells was investigated. TTLL12-overexpressing cell lines were constructed and used to assess the effect of tubulin tyrosine nitration and TTLL12 on the proliferation of SCC-25 cells via western blotting, immunofluorescent and MTT assays. An TTLL12-stably overexpressing SCC-25 cell line and the enzyme-linked immunosorbent assay were used to establish a novel experiment for screening anticancer drugs targeting tubulin tyrosine nitration by assessing its sensitivity, specificity and repeatability, and using it to find an effective drug. The results demonstrated that the proliferative rate of the control cells was notably inhibited in the presence of nitrotyrosine compared with that of TTLL12-overexpressing cells. The results of the MTT assay revealed that the proliferation of TTLL12-silenced cells was significantly inhibited compared with that of the control group. The sensitivity, specificity and repeatability of the experiment were positive. It was found that nocodazole could have better anticancer effect than paclitaxel. Taken together, the results of the present study suggest that TTLL12 enhances SCC-25 cell survival in the presence of nitrotyrosine by disrupting nitration of the tyrosine residues of tubulin, and tubulin tyrosine nitration may be developed for the basic research of anticancer drugs.