Raman microspectroscopy (RM) and polarization sensitive digital holographic imaging (PSDHI) are valuable analytical tools in biological and medical research, allowing the detection of both biochemical and morphological variations of the sample without labels or long sample preparation. Here, using this multi-modal approach we analyze in vitro human sperm capacitation and the acrosome reaction induced by heparin. The multimodal microscopy provides morphofunctional information that can assess the sperms ability to respond to capacitation stimuli (sperm function). More precisely, the birefringence analysis in sperm cells can be used as an indicator of its structural normality. Indeed, digital holography applied for polarization imaging allows for revelation of the polarization state of the sample, showing a total birefringence of the sperm head in non-reacted spermatozoa, and a birefringence localized in the post-acrosomal region in reacted spermatozoa. Additionally, RM allows the detection and spectroscopic characterization of protein/lipid delocalization in the plasma and acrosomal membranes that can be used as valuable Raman biomarkers of sperm function. Interestingly, these spectral variations can be correlated with different time phases of the cell capacitation response. Although further experimentation is required, the proposed multimodal approach could represent a potential label-free diagnostic tool for use in reproductive medicine and the diagnosis of infertility.