While entomophagy is a traditional diet in more than 113 countries, this practice has completely disappeared from the lifestyle in Western countries. However, there is a growing interest in insects as human food in anticipation of the food security challenges related to the worldwide population growth. Nevertheless, food neophobia represents the major challenge which negatively affects the social acceptability for this alternative food resource. It was suggested that the integration of edible insects as food ingredients (protein concentrate or isolate) in different food formulations could enhance the consumer acceptability. In order to do so, an increased knowledge of the nature of the extracted proteins as a function of the food processing used is necessary as it is strongly related to their functional properties. Multiple defatting methods have been explored in the literature since lipid extraction is a key and crucial step during edible insect flours processing as it improves protein recovery and purity. Efficient lipid removal from the solid insect matrix remains conventional Soxhlet extraction by using hexane. However, the use of hexane could have a denaturing effect on edible insects’ proteins resulting in a decrease of their functional properties. Consequently, the aim of this work is to characterize the protein profile of yellow mealworm (Tenebrio molitor) high value-added ingredients according to the defatting process used and to evaluate its impact on protein functional properties. First, high protein content fractions were produced from untreated yellow mealworms with and without an additional hexane defatting step. Their respective protein profiles were compared using 2D-electrophoresis and proteomics. Results showed similar profiles for major edible insect proteins between hexane-defatted and non-hexane-defatted fractions. However, some specific differences were observed in terms of protein species and molecular weights which are under identification by proteomic tools. Our findings can be used to improve insects’ acceptability as an alternate protein source amongst consumers by improving the knowledge surrounding the defatting process during insect meal production and its effect on protein profile and functional properties.