In order to better understand retinal physiology, alterations to which underlie some ocular diseases, we set out to establish the lipid signature of two fundamental cell types in the retina, Müller Glia and Retinal Ganglion Cells (RGCs). Moreover, we compared the lipid signature of these cells in sections (in situ), as well as after culturing the cells and isolating their cell membranes (in vitro). The lipidome of Müller glia and RGCs was analyzed in porcine retinal sections using Matrix Assisted Laser Desorption Ionization Imaging Mass Spectrometry (MALDI-IMS). Isolated membranes, as well as whole cells from primary cell cultures of RGCs and Müller glia, were printed onto glass slides using a non-contact microarrayer (Nano Plotter), and a LTQ-Orbitrap XL analyzer was used to scan the samples in negative ion mode, thereafter identifying the RGCs and Müller cells immunohistochemically. The spectra acquired were aligned and normalized against the total ion current, and a statistical analysis was carried out to select the lipids specific to each cell type in the retinal sections and microarrays. The peaks of interest were identified by MS/MS analysis. A cluster analysis of the MS spectra obtained from the retinal sections identified regions containing RGCs and Müller glia, as confirmed by immunohistochemistry in the same sections. The relative density of certain lipids differed significantly (p-value ≤ 0.05) between the areas containing Müller glia and RGCs. Likewise, different densities of lipids were evident between the RGC and Müller glia cultures in vitro. Finally, a comparative analysis of the lipid profiles in the retinal sections and microarrays identified six peaks that corresponded to a collection of 10 lipids characteristic of retinal cells. These lipids were identified by MS/MS. The analyses performed on the RGC layer of the retina, on RGCs in culture and using cell membrane microarrays of RGCs indicate that the lipid composition of the retina detected in sections is preserved in primary cell cultures. Specific lipid species were found in RGCs and Müller glia, allowing both cell types to be identified by a lipid fingerprint. Further studies into these specific lipids and of their behavior in pathological conditions may well help identify novel therapeutic targets for ocular diseases.