Abstract: The bioassay showed that Gaomi cotton aphid had higher resistance to organophosphate insecticides than Beijing cotton aphid. Using two approaches, a conventional spectrophotometer (method A) and a 96-well microplate reader method (method B),to detect general α-naphthyl acetate(α-NA) esterase activity and kinetics of α--NA esterase of Gaomi and Beijing cotton aphid. The α-NA esterase activity of Beijing and Gaomi cotton aphid were 2.23 and 4.48 (method A),1.28 and 3.30 μmol·mg-1 protein·min-1 (method B),respectively; The α-NA esterase activity ratio of G/B were 2.00 and 2.56; Km of α-NA esterase in Beijing and Gaomi cotton aphid were 6.06×10-5 and 7.51×10-5 (method A),7.66×10-5 and 8.87×10-5 (method B) mol·L-1 , respectively; Vmax were 2.53 and 5.82 (method A),1.28 and 3.61(method B) μmol·mg-1 protein·min-1 , respectively. Data obtained from the 96-well microplate reader were compared to those obtained, under similar conditions, in a conventional spectrophotometer. The approach of using the 96-well microplate reader should be generally applicable to monitor pest resistance to insecticide.