Abstract: A RP-HPLC method was developed to determine the norfloxacin hemolymph concentration of the giant tiger shrimp, Penaeus monodon. HPLC analysis was performed by Agilent 1100. The column was Agilent ZORBAX SB-C18. The mobile phase consisted of acetonitrile:phosphate buffer(10 mmol/L sodium phosphate monobasic and 5 mmol/L tetrabutylammonium bromide)(14.5:85.5,V/V). The flow rate 0.5 mL·min-1, the UV detector wasset at 280 nm, and the column temperature was 25℃. The linear range of norfloxacin was 0.051-20.4 ug/mL, the recovery of norfloxacin from hemolymph was 96.49±1.60%, and theintra-day and inter-day precision were under 3%. The RP-HPLC method could be adapted to determine the norfloxacin concentration in hemolymph and was used to briefly study the pharmaokinetics of norfloxacin in the shrimp. A two-compartment open pharmacokinetic model was used to describe the norfloxacin hemolymph concentration-time profile and the pharmacokinetic model was Co=11.73×e -1.133t+22.62×e-10.918t.The model-derived pharmacokinetic parameters were gained. The distribution half-life(tα1/2) and elimination half-life(tβ1/2) were 0.0635 and 0.612 h, respectively; the area under the concentration versus time curve(AUC), total body clearance(CLs) and apparent volume of distribution(Vd) were 12.425 ug·h/mL?804.83 mL/kg·h and 710.35 mL/kg.