Glial cells support neuronal survival and function by secreting neurotrophic cytokines. Retinal Mueller glial cells (RMG) support retinal neurons, especially photoreceptors. These highly light-sensitive sensory neurons recept vision and their death results in blinding diseases. It has been proposed that RMG release factors that support photoreceptor survival but the nature of these factors remains to be elucidated. To discover such neurotrophic factors, we developed an integrated workflow towards systematic identification of neuroprotective proteins, which are, like most cytokines expressed only in minute amounts. This strategy can be generally applied to identify secreted bioactive molecules from any body fluid once a recipient cell for this activity is known. Towards this goal we first isolated conditioned medium (CM) from primary porcine RMG cultured in vitro and tested for survival-promoting activity using primary photoreceptors. We then developed a large-scale, microplate-based cellular high content assay that allows rapid assessment of primary photoreceptor survival concomitant with biological activity in vitro. The enrichment strategy of bioactive proteins towards their identification consists of several fractionation steps combined with tests for biological function. Here, we combined 1) size fractionation, 2) ion-exchange chromatography, 3) reverse phase liquid chromatography and 4) mass spectrometry (Q-TOF-MSMS or MALDI-MSMS) for protein identification. As a result of this integrated workflow, the insulin-like growth factor binding proteins IGFBP-5 and IGFBP-7 and connective tissue growth factor (CTGF) were identified as likely candidates. Cloning and stable expression of these three candidate factors in HEK293 cells produced conditioned medium enriched for either one of the factors. IGFBP5 and CTGF, but not IGFBP7 significantly increased photoreceptor survival when secreted from HEK cells and when added to the original RMG-CM. This indicates that the survival-promoting activity in RMG-CM is multi-factorial, with IGFBP5 and CTGF as an integral part of this activity.