Bottle bioassay measuring time-mortality rate is a simplified procedure for detecting insecticide resistance. It can be used with biochemical microplate assay to identify mechanism involved. This integrated approach was used to detect temephos resistance in Aedes aegypti from Nonthaburi and Roi Et. Ae. aegypti BKK1 laboratory strain was used as the susceptible reference strain. Appropriate concentration of insecticide for bottle bioassay determined empirically with Ae. aegypti BKK1 strain was found to be in the range of 800-1,050 mg/bottle. Time-mortality rate at 800 mg/bottle was 170±8.66 minutes, significantly different from time-mortality rates at 850, 900, 950 and 1,050 mg/bottle (p=0.008) with 135±5.00, 140±8.66, 135±15.00, and 125±8.66 minutes, respectively. Cutoff concentration selected for resistance detection was 850 mg/bottle. Time-mortality rate of Roi Et strain was 382±26.41 minutes, significantly higher than Nonthaburi (150±25.10 minutes) and BKK1 strain (145±20.49 minutes) at p<0.001. Temephos resistance ratio (RR₁₀₀) in Ae. aegypti Roi Et strain was 2.64-fold higher at lethal time (LT₁₀₀) value than the reference Ae. aegypti BKK1 strain. Mean optical density (O.D.) value from biochemical microplate assay for non-specific esterase of Roi Et strain was higher than the mean O.D. for non-specific esterase of both Nonthaburi and BKK1 strains. Insensitive acetylcholinesterase was not found to be responsible for the resistance in the field-collected mosquitoes. This study suggests that esterase detoxification is the primary cause of resistance in Ae. aegypti population from Roi Et and both bottle bioassay and biochemical microplate assay are proven to be promising tools for initial detection and field surveillance for temephos resistance.