Heparan sulfate proteoglycans (HSPGs) distribution in hair follicle (HF) is classically investigated by conventional histology, biochemical analysis, and immunohistochemistry. In this study, a novel approach is proposed to assess hair histology and HSPG distribution changes in the HF at different phases of the hair growth cycle using infrared spectral imaging (IRSI). Particularly, we were interested in the expression of glypican-1 in different HF compartments and their potential roles during hair shaft growth, as their roles are still poorly understood. The distribution of HSPGs in HFs was probed by IRSI using the absorption region relevant to sulfation as a spectral marker. Findings were supported by Western immunoblotting and immunohistochemistry assays focusing on the glypican-1 expression and distribution in HFs. This study demonstrates the capacity of IRSI to identify the different HF tissue structures and to highlight protein, proteoglycan (PG), glycosaminoglycan (GAG), and sulfated GAG distribution in these structures. The comparison between anagen, catagen, and telogen phases shows the qualitative and/or quantitative evolution of GAGs, as supported by Western immunoblotting. Thus, IRSI can simultaneously reveal the location of proteins, PGs, GAGs and sulfated GAGs in HFs in a reagent- and label-free manner. From a dermatological point of view, IRSI shows its potential as a promising technique to study alopecia.