The female reproductive tract simultaneously guides and selects high-quality sperm using rheotaxis in mammalian species. Sperm quality, however, is traditionally evaluated only by their movement velocities and concentration using computer-assisted sperm analysis (CASA), which ignores sperm rheotaxis. Here, by mimicking the female reproductive tracts' dimensions and hydrodynamic features, a new method is introduced to quantify sperm rheotaxis ability for evaluating semen quality. The combination of our RHEOtaxis quaLity indEX (RHEOLEX) and motile sperm concentration is able to predict sperm fertility levels in artificial insemination at various shear rates within 5 minutes. This means that RHEOLEX could be a biomarker for determining male in vivo fertility, unlike conventional semen quality parameters which fail to provide statistically significant predictions. In addition, a high RHEOLEX is associated with a low DNA fragmentation index (DFI), showing that this new parameter is able to identify low-DFI samples. Not only does this work highlight the importance of rheotaxis in determining male in vivo fertility, but it also provides a solid benchmark for developing fast microfluidic devices for male fertility prediction as well as DFI. Last, the data imply that the female reproductive tract might use rheotaxis to keep sperm with fragmented DNA from reaching the fertilization site.