The current study relates to a Raman spectroscopy-based method for addressing the problem of sex assessment in mammals. A direct method for sex predetermination in animals is based on the X- and Y-bearing sperm cells sorting before insemination. Our Raman spectroscope allows distinguishing and characterizing the difference between X- and Y-bearing sperm cells by detecting and analyzing their Raman spectra in a non-invasive and non-destructive way.