Amylose content (AC) is critical in evaluating starch properties, significantly influencing the food industry and human nutrition. Amylose, though not completely linear, has a unique structure that makes it a key focus in various scientific fields. Understanding its structural and functional properties is essential for its applications in medical, nutritional, and industrial sectors. Accurate determination of AC is vital for effectively processing and using starch-rich products. The choice of AC determination method depends on the specific application and required accuracy and detail. This review summarizes amylose's structural and functional characteristics and recent advancements in qualitative and quantitative AC determination techniques. It also provides insights into future trends and prospects for these technologies, emphasizing the need for more rapid, convenient, accurate, and customizable methods. In conclusion, advancements in amylose determination should enhance accuracy, speed, and ease of use to improve quality control and applications across various sectors while expanding our understanding of amylose and its functionalities. Amylose is a linear or slightly branched polysaccharide composed of α-glucose units linked by α-(1/4) glycosidic bonds. Its structure influences the physicochemical properties of starch, including digestibility, dispersibility, and rheological properties. High amylose content generally correlates with increased resistance to digestion and enhanced mechanical properties, making it valuable in developing new starch-based materials. Amylose inclusion complexes (AICs) have gained interest in culinary applications due to their resistance to enzymatic breakdown, acting as dietary fibers and potential delivery systems for food applications. Qualitative methods, such as the iodine test, are simple and fast, suitable for initial screening. Semi-quantitative methods, like spectrophotometry, provide a better estimate of AC but lack the precision of quantitative methods. Quantitative techniques, including gravimetric procedures, enzymatic tests, and high-performance liquid chromatography (HPLC), are appropriate for in-depth analysis due to their high repeatability, accuracy, and sensitivity. These methods are essential for determining the exact quantity or proportion of amylose in starch samples. Amylose's structural and functional properties are crucial for its applications in food, medicine, and industry. Its unique helical structure and ability to form inclusion complexes make it valuable for various applications, including food packaging, drug delivery, and nutritional benefits. The determination of AC is essential for quality control and developing starch-based products with desired characteristics. Future research should focus on improving the accuracy, speed, and ease of use of AC determination methods to enhance their applications in various sectors.Amylose content (AC) is critical in evaluating starch properties, significantly influencing the food industry and human nutrition. Amylose, though not completely linear, has a unique structure that makes it a key focus in various scientific fields. Understanding its structural and functional properties is essential for its applications in medical, nutritional, and industrial sectors. Accurate determination of AC is vital for effectively processing and using starch-rich products. The choice of AC determination method depends on the specific application and required accuracy and detail. This review summarizes amylose's structural and functional characteristics and recent advancements in qualitative and quantitative AC determination techniques. It also provides insights into future trends and prospects for these technologies, emphasizing the need for more rapid, convenient, accurate, and customizable methods. In conclusion, advancements in amylose determination should enhance accuracy, speed, and ease of use to improve quality control and applications across various sectors while expanding our understanding of amylose and its functionalities. Amylose is a linear or slightly branched polysaccharide composed of α-glucose units linked by α-(1/4) glycosidic bonds. Its structure influences the physicochemical properties of starch, including digestibility, dispersibility, and rheological properties. High amylose content generally correlates with increased resistance to digestion and enhanced mechanical properties, making it valuable in developing new starch-based materials. Amylose inclusion complexes (AICs) have gained interest in culinary applications due to their resistance to enzymatic breakdown, acting as dietary fibers and potential delivery systems for food applications. Qualitative methods, such as the iodine test, are simple and fast, suitable for initial screening. Semi-quantitative methods, like spectrophotometry, provide a better estimate of AC but lack the precision of quantitative methods. Quantitative techniques, including gravimetric procedures, enzymatic tests, and high-performance liquid chromatography (HPLC), are appropriate for in-depth analysis due to their high repeatability, accuracy, and sensitivity. These methods are essential for determining the exact quantity or proportion of amylose in starch samples. Amylose's structural and functional properties are crucial for its applications in food, medicine, and industry. Its unique helical structure and ability to form inclusion complexes make it valuable for various applications, including food packaging, drug delivery, and nutritional benefits. The determination of AC is essential for quality control and developing starch-based products with desired characteristics. Future research should focus on improving the accuracy, speed, and ease of use of AC determination methods to enhance their applications in various sectors.