Objective:To determine the contents of the amino acid neurotransmitters in insomnia rats’ brain tissue. Methods: Copying the drug-induced insomnia rats model,platform in water environment-induced rats model and stimulation-induced rats model,using the HPLC-FD as the testing tool,Agilent ZORBAX SB-Aq (250mm x 4.6mm, 5μm) as chromatographic column which temperature was 25℃,excitation wavelength was 357nm,emission wavelength was 455nm,using methanol-sodium acetate buffer(50mmol/L,pH 6.5) as mobile phase and adopting gradient elution way,to establish the determination methods of the glutamate(Glu), glycine(Gly), γ-aminobutyric(γ-GABA) and taurine (Tau) in the control group rats and the three kinds of insomnia model rats. Results: The standard curve of Glu,Gly,γ-GABA and Tau were linear in 10.06~0.0503,10.13~0.0506,10.05~0.0502,10.03~0.0501μg/mL (the correlation coefficient were 0.99995,0.99995,0.99985 and 0.99990).Contents of Glu,Gly,γ-GABA and Tau in drug-induced insomnic rats were 0.2042?.0145,0.0086?.0005,0.0919?.0024,0.0421?.0011μg. Contents of Glu,Gly,γ-GABA and Tau in platform in water environment-induced insomnic rats were 0.2144?.0159,0.0085?.0004,0.0966?.0035,0.0433?.0012μg.Contents of Glu,Gly,γ-GABA and Tau in stimulation-induced insomnic rats were 0.1818?.0043,0.0084?.0005, 0.0824?.0033,0.0414?.0018μg.While Contents of Glu,Gly,γ-GABA and Tau in control group rats were 0.1744?.0038,0.0085?.0004,0.0791?.0022, 0.0406?.0012μg,respectively n=8.Conclusions:This method can satisfy the demands of the simultaneous determination of Glu,Gly,Tau and γ-GABA in rats’ brain tissue. There may be exist certain relationship among Glu, Tau , γ-GABA and insomnia,the three kinds of insomnia animal model can reflect the changes in amino acid neurotransmitters in the brain well。