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The present study investigated six varieties of locally grown wheat (Lasani, Sehar, Miraj-08, Chakwal-50, Faisalabad-08 and Inqlab) procured from Punjab Seed Corporation, Lahore, Pakistan for their proximate contents. On the basis of protein content and ready availability, Faisalabad-08 (FD-08) was selected to be used for the assessment of comparative efficiency of various methods used for gluten extraction. Three methods, mechanical, chemical and microbiological were used for the extraction of gluten from FD-08. Each method was carried out under ambient conditions using a drying temperature of 55°C. Mechanical method utilized four different processes viz:- dough process, dough batter process, batter process and ethanol washing process using standard 150 mesh. The starch thus obtained was analyzed for its proximate contents. Dough batter process proved to be the most efficient mechanical method and was further investigated using 200 and 300 mesh. Gluten content was determined using sandwich ω-gliadin enzyme-linked immunosorbent assay (ELISA).The results of dough batter process using 200 mesh indicated a starch product with gluten content of 678 ppm. Chemical method indicated high gluten content of more than 5000 ppm and the microbiological method reduced the gluten content from 2500 ppm to 398 ppm. From the results it was observed that no gluten extraction method is viable to produce starch which can fulfill the criteria of a gluten free product (20ppm).