Retinal Müller glial cells (RMG) are recognized as essential players providing neurotrophic, metabolic and structural support for retinal neurons as well as mediating inflammatory responses in the retina. While some key neuroprotective molecules in the context of retinal degeneration, such as FGF2, LIF, PEDF have been demonstrated to be of RMG origin, there is yet no comprehensive understanding on the multifaceted role of RMG in orchestrating diverse intercellular functions. In order to systematically as well as functionally analyse RMG reactivity to retinal degeneration and thus explore the RMG-derived signalling molecules in depth, we combined genomics and proteomics approaches based on profiling primary RMGs from mouse and pig. However, since modulations in cell to cell communication by secretion of molecules may not necessarily present with changes in the mRNA profile, we developed shotgun proteomics approaches enabling direct protein profiling of the RMG lysates and secretomes using label-free and SILAC quantitations. We have identified a pool of novel proteins relevant for pro-survival effects transmitted from RMG to retinal neurons and functionally valdidated selected molecules. Additionally, our approach allows to identify RMG reactions to intrinsic and extrinsic stimuli and thus enables to molecularly dissect RMG reactivity relevant for retinal health and disease.