Soluble amyloid β oligomers (AβOs) are widely recognized neurotoxins that trigger aberrant signaling in specific subsets of neurons, leading to accumulated neuronal damage and memory disorders in Alzheimer's disease (AD). One of the profound downstream consequences of AβO-triggered events is dysregulation of cytosolic calcium concentration ([Ca2+]i), which has been implicated in synaptic failure, cytoskeletal abnormalities, and eventually neuronal death. We have developed an in vitro/in vivo drug screening assay to evaluate putative AβO-blocking candidates by measuring AβO-induced real-time changes in [Ca2+]i. Our screening assay demonstrated that the anti-AβO monoclonal antibody ACU3B3 exhibits potent blocking capability against a broad size range of AβOs. We showed that picomolar concentrations of AβOs were capable of increasing [Ca2+]i in primary neuronal cultures, an effect prevented by ACU3B3. Topical application of 5 nM AβOs onto exposed cortical surfaces also elicited significant calcium elevations in vivo, which was completely abolished by pre-treatment of the brain with 1 ng/mL (6.67 pM) ACU3B3. Our results provide strong support for the utility of this functional screening assay in identifying and confirming the efficacy of AβO-blocking drug candidates such as the human homolog of ACU3B3, which may emerge as the first experimental AD therapeutic to validate the amyloid oligomer hypothesis.