Citrus seeds, a significant by-product of the fruit processing industry, remain an unexploited resource for essential oils with remarkable techno-functional and economic potential. These oils are rich in bioactive compounds such as aldehydes, terpenes, and esters, known for their antioxidant, therapeutic and antimicrobial characteristics, making them valuable in pharmaceutical and food processing sectors. This chapter explores extraction methods, including solvent-based techniques, and highlights advanced analysis tools like gas chromatography (GC), gas chromatography–mass spectrometry (GC–MS), high-performance liquid chromatography (HPLC), and enzyme-linked immunosorbent assay (ELISA). Furthermore, the key precautions ensuring precision and reliability during the extraction and analysis are also discussed. By emphasizing sustainable practices and resource efficiency, this chapter reveals the potential of citrus seed waste as a valuable resource for diverse industrial applications. Hence, this approach not only promotes waste valorization but also contributes to economic and environmental sustainability in the food processing sector.