Cotton fiber in the U.S. is classified for color with the Uster® 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 spectrophotometers. For cotton fiber measurements, glass placed between the sample and the spectrophotometer measurement port is recommended. 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) determine the feasibility of minimizing the major impacts; and 3) develop uniform protocols for measuring cotton fiber on spectrophotometers. Evaluations were made on AMS standard tiles, AMS cotton biscuits, and loose cotton samples. The major impact on spectrophotometer color results was glass use, with the glass impact increasing with increasing glass thickness. L* was the color parameter most impacted by glass use, specular component, and applied fiber pressure. The optimum applied pressure for color measurements on small portions of loose cotton fibers was 30 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, glass with glass calibration, and constant applied pressure for fiber measurements.