Skin fibroblasts are recognized as a valuable model of primary human cells able of mirroring the chronological and biological aging. Here, a lipidomic study of glycosphingolipids (GSL) occurring in the easily accessible human dermal fibroblasts (HDF) is presented. Reversed-phase liquid chromatography with negative electrospray ionization (RPLC-ESI) coupled to either orbitrap or linear ion-trap multiple-stage mass spectrometry was applied to characterize GSL in commercially adult and neonatal primary human fibroblast cells and in skin samples taken from an adult volunteer. Collision-induced dissociation in negative ion mode allowed us to get information on the monosaccharide number and ceramide composition, whereas tandem mass spectra on the ceramide anion was useful to identify the sphingoid base. Nearly sixty endogenous GSL species were successfully recognized, namely 33 hexosyl-ceramides (i.e., HexCer, Hex2Cer and Hex3Cer) and 24 gangliosides as monosialic acid GM1, GM2 and GM3, along with 5 globosides Gb4. An average content of GSLs was attained and the most representative GSL in skin fibroblasts were Hex3Cer, also known as Gb3Cer, followed by Gb4, HexCer and Hex2Cer , while gangliosides were barely quantifiable. The most abundant GSLs in the examined cell lines share the same ceramide base (i.e. d18:1) and the relative content was d18:1/24:1 > d18:1/24:0 > d18:1/16:0 > d18:1/22:0.