Microglia become activated by disturbances in the homeostasis of their local microenvironment. While there are varying degrees of microglia activation, a pro-inflammatory reactive state induced by exposure to stimuli like LPS and IFN-γ. In this study, using label-free PRM approach which is allowing us to measure the hyper-multiplex quantitative value of each protein with high-resolution mass spectrometry, we have quantified over 450 peptides at single run corresponding with the proteins belong to the pathways and directly or indirectly interacted proteins based on STRING database.Moreover, we performed longitudinal quantification of secreted proteins during the detrimental activation of microglia which releases neurotoxic molecules mediated neuronal cell loss in the brain region.