Thiopurines (TPRs) are effective drugs in treating neuromyelitis optica spectrum disorders (NMOSD) and other diseases. TPRs’ toxicity is mainly imputed to thiopurine S-methyltransferase (TPMT) activity. In Chinese population, the most common and important variation of TPMT is TPMT*3C (rs1142345). This study aims to reveal the association between TPMT activity and genetic polymorphisms of TPMT in patients with NMOSD in China. An LC–MS/MS method was used to evaluate the TPMT activity by using 6-mercapthioprine as substrate in human erythrocyte hemolysate via 1 h incubation at 37 °C to form its methylated product 6-methylmercaptopurine (6-MMP). The amount of 6-MMP was adjusted by hematocrit and normalized to 8 × 108 erythrocytes. The selected polymorphisms of TPMT were identified using MassARRAY system (Sequenom) and multiple SNaPshot technique. In 69 patients with NMOSD, TPMT activity was 80.29–154.53 (127.51 ± 16.83) pmol/h/8 × 108 erythrocytes. TPMT*3C (rs1142345) was associated with a lower TPMT activity (BETA = −25.37, P = 0.011). Other selected variants were not associated with the TPMT activity. Further studies are warranted to confirm the results. © 2018 Elsevier Ltd