High-throughput transcriptomics and proteomics approaches have recently identified a large number of germ cell-specific genes with many that remain to be studied through functional genetics approaches. Serine proteases (PRSS) constitute nearly one-third of all proteases, and, in our bioinformatics screens, we identified many that are testis specific. In this study, we chose to focus on Prss44, Prss46, and Prss54, which we confirmed as testis specific in mouse and human. Based on the analysis of developmental expression in the mouse, expression of all four genes is restricted to the late stage of spermatogenesis concomitant with a potential functional role in spermiogenesis, spermiation, or sperm function. To best understand the male reproductive requirement and functional roles of these serine proteases, each gene was individually ablated by CRISPR/Cas9-mediated ES cell or zygote approach. Homozygous deletion mutants for each gene were obtained and analyzed for phenotypic changes. Analyses of testis weights, testis and epididymis histology, sperm morphology, and fertility revealed no significant differences in Prss44, Prss46, and Prss54 knockout mice in comparison to controls. Our results thereby demonstrate that these genes are not required for normal fertility in mice, although do not preclude the possibility that these genes may function in a redundant manner. Elucidating the individual functional requirement or lack thereof of these novel genes is necessary to build a better understanding of the factors underlying spermatogenesis and sperm maturation, which has implications in understanding the etiology of male infertility and the development of male contraceptives.