Technical Abstract: The Uster® High Volume Instrument (HVI) is used to class U.S. cotton for fiber color, yielding the industry accepted, cotton-specific color parameters Rd and +b. The HVI examines a 9 square inch fiber sample, and it is also used to test large AMS standard cotton “biscuits” or rectangles. Much interest has been shown in measuring cotton color on modern color spectrophotometers and on relating the HVI Rd and +b color parameters to more globally recognized, 3-dimensional color space systems obtained from spectrophotometers, such as CIELAB or L*a*b*. Recent research has demonstrated and validated the relationships of Rd and +b to L*a*b*. However, concerns have been expressed on the consistency of fiber sampling/measurement of large volumes of cotton fiber on spectrophotometers. When cotton fiber is measured for color on color spectrophotometers, hand pressure by an operator is often used to hold the fiber against the spectrophotometer sampling port for the color measurement, but the pressure applied varies from operator to operator and from sample to sample. The need exists for a spectrophotometer fiber sampling system for large area fiber samples that allows for the application of uniform pressure to large areas of cotton fiber samples. A program was implemented to develop a pressurized fiber sampling system for color measurements with large cotton fiber samples (e.g., AMS standard cotton “biscuits”). A sampling system for large cotton fiber samples was developed for color spectrophotometers, in which a uniform pressure is applied across the entire large area sample. The color parameter most impacted by fiber pressure was L*. Protocols were developed for pressurized cotton fiber color measurements on spectrophotometers.