Mosquito are major vectors, considering the havoc they play by transmitting many diseases which have greatly affected human beings worldwide. The ecological, socioeconomic conditions of different regions and the emergence of insecticide resistance in main vectors are strongly responsible for determining the geographical distributions and incidence of vector-borne diseases. Among mosquitoes, Anopheles species are responsible for transmission of filariasis, Japanese encephalitis virus and malaria in subtropical region. An. subpictus is a confirmed vector in many countries of South and Southeast Asia. The present study was designed due to limited earlier records regarding insecticide susceptibility status of An. subpictus from district Kasur, Punjab. During the first half of the study, three insecticides i.e. DDT 4%, deltamethrin 0.05% and permethrin 0.75% were testing by using WHO susceptibility bioassay. A total approximately (n=1000) different types of mosquitoes were caught from district Kasur. Mosquitoes belonging to three genra Anopheles, Culex and Aedes were found in our collection. After species identification, An. subpictus was separated for further processing. It was highly abundant species among Anophelines species captured. Field collected adult blood fed females of An. subpictus was reared in the insectary for F1 generation. Two to three days old non engorged adults of An. subpictus were evaluated by using WHO susceptibility bioassays. Both male and female mosquitoes have shown resistance against all three insecticides tested. Probit analysis was used to check the time-response values. Percentage mortalities were recorded against DDT, deltamethrin and permethrin (mortality range 29.47 %-76.28 %.) with higher KT50 and KT95 values.Second half of the study was undertaken to quantify detoxifying enzymes in An. subpictus following WHO biochmical assays. Biochemical analysis of detoxifying enzymes in An. subpictus has revealed that there is significant alteration/elevation of metabolic enzymes when compared with the lab strain might be a contributing factor in conferring insecticides resistance. Elevated levels of GST (0.469 ± 0.115), MFOs (11.665 ± 4.165), ? esterases (1.5808 ± 0.7657) and ? esterase (3.9682 ± 2.311) were detected in An. subpictus. There was a significant high alteration of AChE enzyme activity detected in this species with 57.52% (± 9.234) mean percentage propoxur inhibition. These enzymes are implicated in the metabolism of DDT, pyrethroids and carbamate insecticides.