The hair follicle dermal papilla is composed primarily of extracellular matrix (ECM) proteins secreted by resident fibroblasts. Dermal papilla is endowed with hair morphogenic properties, yet its composition is poorly characterized. In an attempt to understand its specificity better, we compared the protein composition of ECM secreted by cultured dermal papilla fibroblasts with that of dermal fibroblasts. ECM proteins are generally large, difficult to solubilize, and abundantly post-translationally modified. We thus implemented an original protocol for analyzing them: ECM samples were enzymatically digested directly in the culture flasks and analyzed by LC-MS/MS. Sequencing of proteolytic peptides by MS/MS yielded protein identification. The relative abundance of a given protein in dermal fibroblast versus dermal papilla samples was estimated by comparing proteolytic peptide intensities detected by MS. Using this approach, several matrix proteins were found to be present at markedly different levels in each ECM type; in particular, thrombospondin 1 and fibronectin appeared to be overrepresented in the dermal papilla fibroblast ECM. MS results were supported by Western blot and immunostaining experiments. In addition, peptide intensities were processed in two ways, which proved to favor either the quantification accuracy or the information precision at the sequence level.