Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease primarily affecting motor neurons. Unfortunately, there are only two drugs approved to treat the condition, neither of which increases patient survival by more than a few months. This sobering reality highlights the urgent need for new ALS therapeutic development, which has been plagued by high failure rate of drug candidates during clinical trials. This high failure rate suggests that preclinical screening strategies need to be re-evaluated. One of the markers of disease in ALS patients is the aberrantly low expression of neurofilament light chain (NFL) in motor neurons. Further, recovery of NFL to normal levels prevents hallmark phenotypic changes in ALS neurons. Therefore, we wanted to establish a clinically relevant screening platform to identify compounds that return expression of NFL to normal levels in ALS patient derived motor neurons. At BrainXell, we established new technologies to rapidly differentiate ALS patient induced pluripotent stem cells (iPSCs) into large quantities of neurons. We then used genome editing techniques to endogenously fuse NFL with a nanoluciferase (NLuc) reporter, thus enabling a high-throughput screening (HTS) system that monitors the expression levels of NFL after 72 h exposure to each compound. The assay was adapted to meet HTS requirements, including: large batch sizes, 1536-well format, minimal well-to-well variation, short-term culture, plating by automated dispenser, and low reagent volumes. Applying a quantitative HTS approach, we screened the LOPAC, NPC, and MIPE libraries (>6,000 compounds) in a dose dependent manner. Compounds that increase NFL expression by >30% (to approximately normal levels) were considered hits. From these screens we identified 80 hit compounds that are currently going through secondary validation. Preliminary data look promising. For example, two of these hits restore normal expression of NFL with no observed toxicity.