Here we present a novel 3D bioprinted model of the forebrain cortex designed to quantify neurite outgrowth across a hydrogel bridge. To validate this model, we cultured Alzheimer’s disease (AD) forebrain cortical populations derived from human iPSCs carrying APP (amyloid precursor protein) mutations (K670M/N671L + V717F). Neurite and synapse formation were significantly impaired in 3D AD mutant cultures compared to controls, but this was not replicated in 2D, highlighting deficits in these traditional 2D cell culture models. To investigate the mechanisms underlying impaired neurite outgrowth in 3D and 2D models of AD, we assessed amyloid-β dysfunction, mitochondrial health, and oxidative stress in both conditions. In the 3D model, APP mutant cultures exhibited reduced mitochondrial membrane potential and fragmented networks, indicating dysfunction and potential cellular energy deficits. Additionally, elevated oxidative stress and proteostasis disruption were identified in the 3D AD models as indicators of cellular damage which may be limiting neurite extension. Furthermore, transcriptomic (bulk RNA-Seq) analysis revealed distinct differences in gene expression pathways between 2D and 3D models of AD, suggesting alternate underlying mechanisms of disease pathology between the culture conditions. This study demonstrates the functionality of this novel 3D bioprinted model for quantifying neurite connectivity and in identifying underlying disease mechanisms.