OBJECTIVE：To establish a method for concentration determination of anlotinib in human plasma and apply it in the clinic. METHODS ：The plasma samples were pretreated by salting-out assisted with liquid-liquid extraction with ammonium acetate as salting out assistant and acetonitrile as solvent. Using voriconazole as internal standard ，LC-MS/MS method was adopted. The separation was performed on Waters X Bridge C 18 column with mobile phase consisting of 0.2％ formic acid solution- acetonitrile（gradient elution ）at the flow rate of 1 mL/min. The column temperature was set at 40 ℃，and sample size was 10 μL. The split ratio was 3∶7. The electrospray ion source and multiple reaction monitoring mode were used for the analysis. The ion pair of anlotinib and internal standard under positive ion mode were m/z 408.3→339.3 and m/z 350.2→281.3，respectively. RESULTS ： Anlotinib showed a good linear relationship in the concentration range of 0.2-200 ng/mL（R2＞0.996 7）. The lowest limit of quantitation was 0.2 ng/mL. Intra-day and inter-day RSDs were no more than 12％ （n＝6 or n＝3）. Accuracies were 90.92％-108.00％（n＝6 or n＝3）. The average extraction recoveries were 87.51％-100.00％（RSD＜8％，n＝6）. The average matrix effects were 96.66％-99.93％（RSD＜5％，n＝6）. The plasma concentration of 3 patients with NSCLC treated with anlotinib was 8.74-65.60 ng/mL. CONCLUSIONS ：The method is simple ，accurate and specific ，and is suitable for the plasma concentration monitoring of anlotinib in NSCLC patients.