Hesperidin is a bioflavonoid occurring in high concentrations in citrus fruits. Its use has been associated with a great number of health benefits, including antioxidant, antibacterial, antimicrobial, anti-inflammatory and anticarcinogenic properties. The food industry uses large quantities of citrus fruit, especially for the production of juice. It results in the accumulation of huge amounts of by-products such as peels, seeds, cell and membrane residues, which are also a good source of hesperidin. Thus, its extraction from these by-products has attracted considerable scientific interest with aim to use as natural antioxidants. In this review, the extraction and determination methods for quantification of hesperidin in fruits and by-products are presented and discussed as well as its stability and biological activities.