Human induced pluripotent stem cell-derived (iPSC) neural cultures offer clinically relevant models of human diseases, including Amyotrophic Lateral Sclerosis, Alzheimer’s, and Autism Spectrum Disorder. In situ characterization of the spatial-temporal evolution of cell state in 2D and 3D culture models and organoids based on protein expression levels and localizations is essential to understanding neural cell differentiation, disease state phenotypes, and sample-to-sample variability. Here we apply PRobe-based Imaging for Sequential Multiplexing (PRISM) to facilitate multiplexed imaging with facile, rapid exchange of imaging probes to analyze iPSC-derived cortical and motor neuron cultures that are relevant to psychiatric and neurodegenerative disease models, using over ten protein targets. Our approach permits analysis of cell differentiation, cell composition, and functional marker expression in both standard 2D cultures and 3D spheroid and organoid sections. Further, our approach is amenable to automation, offering in principle ability to scale-up to dozens of protein targets and samples.