Maturity and fineness are important physical properties of cotton fibers affecting qualities of fibers and yarns. A number of methods are used for measuring fiber maturity and fineness from developed fibers that are desiccated and harvested from open bolls. With the recent advent of molecular breeding and genomics, there is a growing need for measuring the physical properties of developing fibers that are living cells containing genetic materials. Unlike the developed fibers, the developing fibers are immature fibers composed of high levels of physiological sugars causing stickiness. Therefore, there is a challenge in measuring fiber properties from the developing fibers. To identify methods enabling the measurement fiber maturity and fineness from developing fibers, we compared various methods including the use of the USTER Advanced Fiber Information System (AFIS), high-volume instrument, Cottonscope, fiber cross-section image analysis microscopy, cellulose assay, and gravimetric fineness methods. Our results showed that maturity ratios (MR) measured from high maturity fibers correlated among all methods, whereas AFIS MR measured from low maturity fibers did not correlate with other methods. The fineness values measured by AFIS and Cottonscope were affected by the levels of physiological sugars in developing fibers. As a result, we conclude that pre-elimination of physiological sugars causing the stickiness was crucial to measure accurate fineness values from developing fibers. The results demonstrated strengths and weaknesses of various methods of measuring fiber maturity and fineness from immature and developing fibers. The information will help cotton scientists measuring and interpreting fiber properties from developing fibers.