Technical Abstract: Cotton production and usage is a global enterprise, and the export of U.S. cotton has increased dramatically. In the U.S., cotton is classed (and its primary quality parameters determined) by the Uster® High Volume Instrument (HVI), which must be maintained under tightly controlled laboratory environmental conditions. For U.S. cotton to successfully compete in the global marketplace, improved and fast response quality measurement systems and tools are needed to rapidly assess the quality of cotton. One key area of emphasis and need is the development and implementation of new fast-response quality measurements that can be used not only in the laboratory but which also can be adapted to field and at-line quality measurements. Many advances have been made in the area of portable spectroscopic instrumentation, and these advances may lead to improved, rapid, and precise quality tools for laboratory and breeder analyses. A program was implemented to determine the ability of advanced portable instrumentation and techniques to monitor critical fiber properties of cotton samples in the laboratory, at-line, and in the field. Initial emphasis was on portable Near InfraRed (NIR) instruments and the measurement of cotton fiber micronaire in the laboratory. Micronaire is a key cotton property, and it is an indicator of the fiber’s maturity and fineness. Spectral evaluations demonstrated very good NIR spectral agreement between portable and bench-top NIR units for micronaire. Distinct NIR spectral differences between samples with varying micronaire were observed. In this paper, a comparative evaluation was performed to determine optimum instrumental and operational conditions and settings for cotton fiber micronaire measurements. The comparative evaluation established that the optimum instrumental conditions for laboratory measurements of fiber micronaire was obtained with the use of a glass-covered sampling port and increased instrumental gain. Very good validation results were obtained with the optimal condition, with high R2, low residuals, and with = 12% outliers. For a NIR measurement with potential for multiple simultaneous analyses and non-laboratory measurements, the micronaire measurement was fast (< 3 minutes per sample) and easy to perform. The rapid and accurate laboratory measurement of cotton fiber micronaire with portable NIR instrumentation was demonstrated.