Protein extraction from wheat cultivars (cvs) and aphids was refined through trichloroacetic acid, phenol extraction, and dye-lysis methods. In this study, two wheat cultivars (Galaxy 2013 and NARC 449) were evaluated for their interaction with Sitobion avenae and Rhopalosiphum padi. Aphids were collected from wheat fields and reared on the wheat cultivars under controlled conditions. Protein extraction from both wheat cultivars and aphids employed by three distinct methods: TCA–acetone extraction, phenol extraction, and the Dye-Lysis buffer method. The Dye-Lysis buffer method demonstrated superior consistency and reproducibility in protein band patterns compared to the other extraction protocols. Protein expression variations in aphids and wheat cultivars pre- and post-aphid feeding were identified using Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis. Densitometry analysis was utilized for the precise quantitation of differentially expressed proteins. Comparative proteomic analysis of wheat cultivars under aphid stress revealed differential expression of proteins related to defence mechanisms, photosynthetic proteins and stress-responsive proteins. Key photosynthetic proteins such as Protoporphyrin IX magnesium chelatase and RuBisCO exhibited downregulation, while stress-responsive proteins like heat shock proteins, as well as defense-related proteins displayed up-regulation following aphids’ infestation. Aphids, specifically S. avenae and R. padi, under infestation conditions, exhibit elevated concentrations of yellow e-3-like protein, peroxidase, trypsin, carbonic anhydrase, apolipophorins, and glyceraldehyde-3 phosphate dehydrogenase in comparison to non-feeding counterparts. These findings contribute to a deeper understanding of the molecular interactions between wheat plants and aphids, providing insights into potential defense mechanisms and vulnerabilities in the host-pathogen relationship.