Fruit ripening is a key developmental phase with biochemical and physiological changes. In climacteric fruits (ethylene responsive), such as bananas, ethylene predominantly regulates these changes. This study presents a protocol to isolate and functionally analyse proximal promoters of banana ripening genes. Using genome walking, we identify promoter fragments of key ripening genes. We then characterize their ethylene responsiveness. Promoter–GUS fusion constructs are prepared for functional testing. We deliver these constructs transiently into banana fruit tissues. Promoter activity is quantified by histochemical (color-based) and fluorometric (fluorescence-based) β-glucuronidase (GUS) assays. Findings clarify the regulation of ripening-associated pathways. Therefore, they suggest genetic engineering options to extend shelf life and improve banana nutritional quality. This protocol provides a practical guide for researchers to study and manipulate the molecular mechanisms underlying fruit ripening.