The expression of glypicans in different hair follicle (HF) compartments is still poorly understood. Heparan sulfate proteoglycans (HSPGs) distribution in HF is classically investigated by conventional histology, biochemical analysis, and immunohistochemistry. Our previous study proposed a novel approach to assess hair histology and glypican-1 (GPC1) distribution changes in the HF at different phases of the hair growth cycle using infrared spectral imaging (IRSI). We show in the present manuscript for the first time complementary data on the distribution of glypican-4 (GPC4) and glypican-6 (GPC6) in HF at different phases of the hair growth cycle using IR imaging. Findings were supported by Western blot assays focusing on the GPC4 and GPC6 expression in HFs. Like all proteoglycan features, the glypicans are characterized by a core protein to which sulfated and/or unsulfated glycosaminoglycan (GAG) chains are covalently linked. Our study demonstrates the capacity of IRSI to identify the different HF tissue structures and to highlight protein, proteoglycan (PG), 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 blot. Thus, in one analysis, IRSI can simultaneously reveal the location of proteins, PGs, GAGs and sulfated GAGs in HFs in a chemical and label-free manner. From a dermatological point of view, IRSI may constitute a promising technique to study alopecia.