The Cucurbitaceae family, known as the gourd family, includes several economically and nutritionally important crops, while the genera Citrullus, Cucumis, and Cucurbita are among the world's top ten economically important crops. Beyond their nutritional benefits, many Cucurbitaceae fruits offer potential health benefits, including antioxidant, antidiabetic, antihypertensive, and antimicrobial properties. However, most studies focus on secondary metabolites instead of bioactive peptides (BPs) – short chains of amino acids – leaving a significant gap in understanding their important role in these crops. Our study will investigate the tissue-specific distribution of BPs in edible and non-edible parts of selected Cucurbitaceae crops and examine the impact of storage conditions and durations on BP abundance. As the preliminary step in identifying and characterising cucurbit BPs, we conducted a genome-guided prediction and identified bioactive motifs from selected cucurbits’ proteomes using an in-house BP database. Cucurbita moschata was identified with the highest BP count per proteome size (24.1), followed by Cucurbita pepo (20.4) and Cucurbita maxima (20.2). Most predicted cucurbit BPs were antimicrobial, followed by antihypertensive, anticancer, antidiabetic, and antioxidant. As the next step, we performed protein extraction using different extraction solvents (deionised water, 1M acetic acid, Tris-HCl, urea, and phenol) to optimise the process for different tissues (seed, peel and flesh) of a few selected Cucurbitaceae crops. The efficiency assessment of these protein extraction solvents and further BP discovery and quantitation will be performed using liquid chromatography-tandem mass spectrometry, a peptidomics approach combined with in-silico data mining, and in-vitro assays for functional characterisation and validation of the identified peptides. This research will provide insights into the tissue-specific abundance and distribution of BPs of the selected Cucurbitaceae crops, which will guide us to explore the potential of valorising processing by-products to develop value-added nutritional and nutraceutical ingredients, enhancing waste management and economic value of these Cucurbitaceae crops.