Alzheimer disease (AD) is characterized by brain depositions of the beta amyloid (Beta-A). The Beta-A is the amyloid precursor protein (APP) digestion product, which is released from the cell after Beta–secretase and Gamma-secretase proteolysis. A novel recombinant cell line has been developed for screening inhibitors for Gamma-secretase activity via APP processing pathway.Cell Line Characterization: APP-C99 U2Os cells allow to perform a Betaays to evaluate the endogenous Gamma-secretase proteolityc process in living cells. This cell line has been validated using different inhibitory compounds for Gamma-secretase (positive controls) and Beta-secretase activity (negative controls). High content analysis of Gamma-secretase activity has been designed to be performed using an epifluorescent imaging system to acquire and analyze images and to quantify the fluorescent vesicles into the citoplasm.The results obtained during the assay validation indicate that the pharmacological inhibition of Gamma-secretase implicated in AD is a valid strategy for drug screening and these models are appropriate to monitor the disease process in vivo in bioimaging systems.Assay Details: This model permits evaluate a library of compounds, candidates to inhibitors, in living cells studying the vesicles retentionThis model provide a strategy to evaluate drug against gamma secretases activity without the necessity to be permeable.This model allows to analyse in the space and time the compound effect in a multiparametric manner.