【Objective】 Cloud point extraction （CPE） combined with hydrophilic interaction （HILIC） was employed to simultaneously determine the anti-AIDS drugs in the serum of rats. 【Methods】Triton X-114  was used as extraction medium to extract four nucleoside antiviral drugs from rat serum. Response surface methodology was employed to further optimize CPE parameters. The content of four anti-AIDS drugs in rat serum was simultaneously determined by HILIC method.【Results】 The optimized conditions were as follows：5%（w/v）Triton X- 114，0.3 mol/L NaCl，PH 5.0， the water- bath equilibrium 20 min at 40 ℃ . Under optimized extraction conditions，the extraction rates of the four anti- AIDS drugs were all over 85.0%，which  was pretty close to the predicted result. The extracted samples were analyzed under the optimal chromatographic conditions. The recovery rate was over 75.0% and RSD was less than 5.0%. The optimized chromatographic conditions were as follows：Ze month- CN（250 × 4.6 mm，5 micron）as stationary phase，mobile phase （methanol：acetonitrile：ammonium acetate buffer）（5∶5∶90），column temperature 35 ℃，detection wavelength 275 nm，flow rate 0.5 mL/min.【Conclusion】The developed method of CPE combined with HILIC can enrich four anti-AIDS drugs in rat serum ，which is simple，environment- friendly and can be used to accurately determine the blood concentration of four anti-AIDS drugs in rat serum，providing a new method for clinical blood concentration monitoring.