The sex of the offspring can be predetermined by flow cytometric sorting of highly purified X- or Y-chromosome-bearing sperm subpopulations. The technology has undergone several improvements in the past 10 years and it is now widely used by dairy and beef cattle, small ruminants, deer, equine and porcine industries. To ensure the quality of the sorted product, Sexing Technologies analyzes various sperm parameters at multiple checkpoints during the sex sorting process. One of these quality parameters is sperm sex purity, which is assessed using a Genesis-1 high resolution Analyzer, a modified MoFlo SX sperm sorter, developed for sperm analysis using ST proprietary technologies. Each Analyzer is calibrated using certified sperm nuclei as a standard, and has to meet predetermined specifications of number of oriented cells for the Side Fluorescence detector path and quality of resolution on DNA content on the Forward Fluorescence detector path before running any purity analysis. This confirms the instrument is well aligned and meeting the required orientation and resolution criteria to perform accurate assessment of sex sorted sperm purity. High performance sperm sorting systems known as Genesis-III have advanced digital processing, multiple heads and automation built in. These systems are verified multiple times a day to confirm that the setup is accurate for the desired sex ratio, while optimizing and maintaining productivity. Estimation of the percent of X- and Y- chromosome-bearing sperm is also performed on each batch of sex-sorted sperm produced. Typically, 3,000–5,000 cells are analyzed per sample depending on the target purity. The implementation of these technological advancements provides high confidence in the quality assessment of Sexing Technologies’ sex-sorted products, which reflects in the most recent field results. Field data collected in the years of 2016–2018 confirmed that an in vitro analyzed X-chromosome-bearing sperm purity of 91.1 ± 0.18% resulted in an in vivo female calf percent of 90.3 ± 0.18%, based on 9,447 calvings.