Despite widespread use of methods to assess structure and functional activity of spermatozoa, practical application of those in in vitro fertilization programs is currently rather limited. Limitations are primarily due to destructive nature of the methods. The study aimed to investigate the capabilities of confocal Raman spectroscopy in analysis of the human sperm organelle biochemical markers. Assessment of 176 spectra of spermatozoa collected from healthy sperm donors aged 18–35 years was performed using the Bruker Senterra confocal Raman microscope (Germany). Spectra were acquired from the sperm acrosome, nucleus, and midpiece. In addition, the spermatozoa suspension was exposed to a focused x-ray beam. As a result, bands were identified inherent to the sperm nuclear DNA  — 1092 cm^–1 and 780 cm^–1, typical for the head — 748 cm^–1 (mitochondrial DNA marker); changes of shape of the triple band 420 cm^–1, 1445 cm^–1 and 1486 cm^–1 with predominance of the middle part 1445 cm^–1 are typical for acrosomal spectra. No differences in the main Raman bands nherent to cells post DNA damage under exposure to x-ray radiation for 5 and 10 min relative to intact samples were reported. Confocal Raman spectroscopy is a promising noninvasive method to assess sperm ultrasctructure and biochemical processes.