Objective To establish an ultra high performance liquid chromatography-tandem quadrupole mass spectrometry (UPLC-MS/MS) method for simultaneous determination of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in cerebrospinal fluid of depressive rats, and to investigate the concentrations of 5-HT and 5-HIAA in cerebrospinal fluid of depression rats and fluoxetine treated rats. Methods Twenty-four SD rats were randomly divided into control group, model group and fluoxetine group, with 8 rats in each group. At 30 d of model building, the cerebrospinal fluids were taken after the test of behaviors, and the concentrations of 5-HT and 5-HIAA were determined by UPLC-MS/MS after pretreatment of protein precipitation. Chromatographic column was ACE C₁₈-PFP column (4.6 mm×150 mm, 3 μm), mobile phase was acetonitrile and 0.2% formic acid-water solution, flow rate was 0.4 mL/min, and the column temperature was 25℃. The mass spectrometry detection was performed using dynamic multiple reaction monitoring (DMRM). Results 5-HT and 5-HIAA in cerebrospinal fluid of depression rats had good linear relationship within the concentration range of 0.10-100.00 ng/mL and 1.00-1 000.00 ng/mL, respectively (r>0.999). Intra-and inter-day precision was less than 15.00%; the recoveries ranged from 86.86% to 98.49%; and the matrix effects ranged from 89.80% to 95.25%. The samples remained stable during analysis. Compared with the control group, the concentration of 5-HT in cerebrospinal fluid of rats in the model group was significantly decreased (P<0.01), and the concentration of 5-HIAA was significantly increased (P<0.01). Compared with the model group, the concentration of 5-HT in cerebrospinal fluid of rats in the fluoxetine group was significantly increased (P<0.01), and the concentration of 5-HIAA was significantly decreased (P<0.01). Conclusion The UPLC-MS/MS method is simple, accurate, sensitive, reproducible and suitable for simultaneous determination of 5-HT and 5-HIAA in cerebrospinal fluid of depression rats.