Synaptic loss represents one of the earliest signs of neuronal damage and is observed within both Alzheimer's disease patients and transgenic mouse models of the disease. We have developed a novel in vitro assay using high content screening technology to measure changes in a number of cell physiological parameters simultaneously within a neuronal population. Using Hoechst-33342 to label nuclei, betaIII-tubulin as a neuron-specific marker, and synapsin-I as an indicator of pre-synaptic sites, we have designed software to interrogate triple-labelled images, counting only those synaptic puncta associated with tubulin-positive structures. Here we demonstrate that addition of amyloid beta peptide (Abeta(1-42)), to either primary hippocampal or cortical neurons for 4 days in vitro has deleterious effects upon synapse formation, neurite outgrowth and arborisation in a concentration-dependent manner. Control reverse peptide showed no effect over the same concentration range. The effects of Abeta(1-42) were inhibited by D-KLVFFA, which contains residues 16-20 of Abeta that function as a self-recognition element during Abeta assembly and bind to the homologous region of Abeta and block its oligomerisation. These effects of Abeta(1-42) on synapse number and neurite outgrowth are similar to those described within AD patient pathology and transgenic mouse models.