Microfibers are small fiber particles that separate from larger textiles through wear abrasion or home laundering. Pervasive accumulation of synthetic microfibers in the environment is motivating efforts to quantify them, and to gain a better understanding of the factors that lead to their release from garments. Automated imaging systems have been previously employed for the quantification of synthetic and natural microfibers. In the current study, a rayon standard and microfibers sourced from scoured cotton HVI calibration standards were examined with the Fiber Quality Analyzer-360 (FQA) automated imaging system. Mechanically stirred suspensions of six cotton microfiber standards showed significantly lower fiber counts than those obtained with a rayon standard. Probe sonication of the sample suspensions significantly increased observed fiber counts for the cotton standard samples, by 105% on average. Mean length determinations decreased by, on average, 5% for the sonicated samples, an indication that count increases were not due to sample fragmentation. No significant change was observed for the fiber counts or length measurements of the sonicated rayon samples. The sonicated cotton samples showed an average of 95% detection by the FQA. These results highlight the importance of proper microfiber suspension for accurate detection and quantification using the FQA system.