Placenta mesenchymal stem cell (PMSC) treatment has been investigated to repair the spina bifida defect in various animal models, including in an English Bulldog model and in the gold-standard fetal lamb model of spina bifida. Furthermore, these cells are currently undergoing a Phase 1/2a clinical trial to assess their safety and preliminary efficacy in humans. For these cells to move forward into Phase 2 and Phase 3 clinical trials, Biologics License Applications (BLA) and commercialization as a biological drug, they must be manufactured and characterized according to FDA guidelines and must be validated through potency assay(s). An industrial standard potency assay must contain replicates of the reference standard, samples, and positive and/or negative controls. The replicates must be in triplicate and serially diluted into 8 or 12 concentrations that would fit down the columns or across the rows of a 96-well plate or a 394-well plate. The range of concentration is determined by the linear range of the assay, which is part of the sample acceptance criteria. The assay must also have a validated assay (system) acceptance criteria and an appropriate analysis method. Currently, an in vitro neuroprotection assay in the 12-well format developed by Kumar et al. has been validated as a screening method for identifying PMSC lines. My thesis is aimed to further refine the parameters and develop a high-throughput, industrial standard potency assay for screening PMSCs. During development, the cell seeding densities for SH-SY5Y and ENStem-A cell line were optimized for the 96-well format. Extracellular matrix coating was also examined for ENStem-A optimal culturing conditions. ImageXpress PICO Automated Cell Imaging System/Neurite Tracing Image Analysis was compared and determined as a better system than the system used for the 12-well format, as this system was easier to use, more accurate, took less time to complete and was automated for high throughput assay. Two possible mechanisms of PMSC protection were elucidated, neurogenesis preservation and neuroprotection. Therefore, two assays were developed and tested with undifferentiated and differentiated SH-SY5Y cells. However, the results were inconclusive, lacking dosage responses. There were differences between the control and the PMSC-rescued samples. There were also differences between the PMSC dosages, but the differences did not produce a linear trend. The discrepancy could be due to the biology of undifferentiated SH-SY5Y as a neuroblastoma line. Plate setting, acquisition time, intensity and image analysis parameters could also be factors accounting for the inconsistency. Further optimization of differentiated SH-SY5Y is needed to achieve 100% mature neurons and specificity for the neuroprotection mechanism. Replacing the live-cell imaging procedure with fixed cells followed by immunocytochemistry (ICC) would preserve cell morphology, prevent cells from lifting off and capture fragile neurite processes by formaldehyde cross-linking, allow multiple acquisitions and analysis with the same sample/plate. Fixed cells/ICC would also aid in establishing the assay acceptance criteria such as plate setting, acquisition time, intensity, and image analysis parameters.