Technical Abstract: A key cotton quality and processing property that is gaining increasing importance is the color of the cotton. Cotton fiber in the U.S. is classified for color using the Uster® High Volume Instrument (HVI), using the parameters Rd and +b. Rd and +b are specific to cotton fiber and are not typical globally recognized color systems. An earlier program established and validated the feasibility of correlating HVI Rd and +b to the globally recognized color system L*a*b*, available on most commercial bench-top and portable spectrophotometers. For cotton fiber measurements, glass placed between the sample and the spectrophotometer measurement port is required. In preliminary evaluations, the major impact on agreement between the HVI and standard color spectrophotometers and between color spectrophotometers was the use of glass. A program was implemented to 1) investigate in detail the impacts of key operational and instrumental parameters on spectrophotometer color measurements applicable to cotton fiber, with emphasis on instrument specular component, glass use, and pressure on the fiber, 2) to determine the feasibility of minimizing the major impacts, and 3) develop uniform protocols for measuring cotton fiber on spectrophotometers. The glass impact on spectrophotometer color results was much greater than that of specular component type, with the glass impact increasing with increasing thickness of the glass used. The color parameter most impacted by glass use, specular component, and applied fiber pressure was L*. The optimum applied pressure for color measurements on small portions of loose cotton fibers was 30 psig (pounds per square inch gauge). Protocols for minimizing glass impacts on spectrophotometer color measurements and for pressurized fiber measurements were determined, with the best overall results obtained with the use of specular component included (SCI), glass with glass calibration, and constant applied pressure for fiber measurements (e.g., 30 psig).