【Abstract】Objective To establish a novel chemotherapy sensitivity assay in vitro with TTLL12 as target. Methods TTLL12 overexpression cell line and Western blot were used to detect the effect of thiirene and paclitaxel on nitrotyrosine microtubulin. The optimal concentration of nitrotyrosine and the drug were determined by TTLL12 overexpression cell line and ELISA. The sensitivity, specificity and repeatability of the novel experimental method were tested. The novel experimental method was used to screen anticancer drugs. The effect of nocodazole on nitrotyrosine microtubulin were detected by Western blot. Results The results of Western blotting showed that nitrotyrosine microtubulin bands were found in paclitaxel group, but no nitrotyrosine microtubulin bands were found in thiirene group. Paclitaxel was identified as a positive control. Thiirene was identified as a negative control. The results of ELISA showed that the concentration of nitrotyrosine was 400uM and the concentration of drug to be tested was 10uM. The sensitivity, specificity and repeatability of the novel experimental method were good. Nocodazole was screened out as an effective anticancer drugs.The results of western showed nocodazole increased nitrotyrosination and suppressed the function of TTLL12. ConclusionThe novel experimental method is simple, fast, stable and sensitive for the screening of anticancer drugs，which has good application value for the basic research of anticancer drugs.