Starch is composed mainly of two polymers, amylose and amylopectin, which show distinct structures. Amylose is essentially a linear polymer with a lower size, which is formed by α-1–4 glycosidic bonds with few α-1–6 branching points, and this polymer shows a molecular mass between 105 and 106 g mol−1. Amylopectin is a branched polymer with a larger size, which is formed by α-1–4 glycosidic bonds with a large number of α-1–6 bonds, and this polymer shows a molecular weight from 107 to 109 g mol−1. These structural differences have an important effect on the characteristics of starch, such as texture, solubility, viscosity, stability, gelatinization, moisture retention, retrogradation, and syneresis, which are the main criteria of selecting an appropriate starch for food products. Moreover, modification processes can partially depolymerize the molecules or promote crosslinking, thus affecting the starch molecular size distribution. In this context, the characterization of the molecular size distribution of starches is important for establishing structure-processing-property relationships, for improvements in human and animal nutrition and industrial applications. In this chapter, we describe the methodology for determining the molecular size distribution of starch using gel permeation chromatography (GPC). GPC separates the starch polymers considering their sizes, which are quantified by separating fractions and using the blue value and/ or the total carbohydrates methods.