Objective To determine dopamine and its metabolites during in vivo cerebral microdialysis by routine high performance liquid chromatography with electrochemical detection. Methods Microdialysis probes were placed into the right striatum of Wistar rat brains and perfused with Ringer's solution at a rate of 1.5 μL/min. A reverse phase HPLC with electrochemistry was used to assay DA, DOPAC, and HVA after cerebral microdialysates were collected every 20 minutes from awake and freely moving rats. In order to identify the reliability of this method, its selectivity, linear range, precision and accuracy were tested and the contents of DA, DOPAC, and HVA in rat microdialysates were determined. Results The standard curve was in good linear at the concentration ranging from 74 nmol/L to 1.5 μmol/L for DOPAC (r2= 0.9996), from 66 nmol/L to 1.3 μmol/L for DA (r2=1.0000) and from 69 nmol/L to 1.4 μmol/L for HVA (r2=0.9992). The recovery of DOPAC (0.30, 0.77, 1.49 μmol/L),DA (0.26, 0.69, 1.32 μmol/L), and HVA (0.27, 0.71, 1.37 μmol/L) was 82.00±1.70%, 104.00±4.00%, 98.70±3.10%;92.30±1.50%, 105.30±2.30%, 108.00±2.00%; 80.00±7.80%, 107.69±8.00%, and 108.66±3.10%, respectively at each concentration. Their intra-day RSD was 3.3%, 3.4%, and 2.5%, and inter-day RSD was 4.2%, 2.3%, and 5.6%, respectively.The mean extracellular concentrations of DOPAC, DA, and HVA in rat brain microdialysates were 10.7, 2.4, and 9.2 μmol/L (n=6), respectively. Conclusion The findings of our study suggested that the simple, accurate and stable method can be applied to basic researches of diseases related to monoamines neurotransmitters by cerebral microdialysis in rats.