The proposed reference method for measuring the maturity of cotton fiber includes procedures for: sampling the cotton; forming a bundle of well-aligned fibers; embedding the bundle in a polymer matrix; cutting a thin cross-section of the bundled fibers; imaging the cross-sections with a light microscope; and using image analysis software to characterize the size, shape, and degree of development (maturity) of the cotton. The Southern Regional Research Center (SRRC) and the International Textile Center (ITC) applied the method to seven bales of specially selected cottons. The maturity results from the two groups were in excellent agreement.