Alzheimer's disease (AD) is a devastating neurodegenerative disease affecting millions of people. The amyloid hypothesis suggests that the pathogenesis of AD is related to the accumulation of amyloid beta (Aβ) in the brain. Herein, the authors quantify Aβ-mediated changes in neuronal morphology in primary cultures using the Cellomics neuronal profiling version 3.5 (NPv3.5) BioApplication. We observed that Aβ caused a 33% decrease in neurite length in primary human cortical cultures after 24 h of treatment compared with control-treated cultures. We also determined that quantifying changes of neuronal morphology was a more sensitive indicator of nonlethal cell injury than traditional cytotoxicity assays. Aβ-mediated neuronal deficits observed in human cortical cultures were also observed in primary rat hippocampal cultures, where we demonstrated that the integrin-blocking antibody, 17E6, completely abrogated Aβ-mediated cytotoxicity. Finally, we showed that Aβ challenge to 21 days in vitro rat hippocampal cultures reduced synapsin staining to 14% of control-treated cultures. These results are consistent with the finding that loss of presynaptic integrity is one of the initial deficits observed in AD. The implementation of phenotypic screens to identify compounds that block Aβ-mediated cytotoxicity in primary neuronal cultures may lead to the development of novel strategies to prevent AD.