Abstract Body: Hypoplastic Left Heart Syndrome (HLHS) is characterized by an underdeveloped left ventricle and aorta. Patients diagnosed with HLHS require surgical intervention in the first week of birth followed by additional surgeries afterwards due to a lack of available nonsurgical treatment options. Mutations in NOTCH1 have been identified in HLHS, although this insight has yet to be translated into therapeutic options. This is due to our limited understanding of the molecular mechanisms which drive both cardiogenesis and HLHS pathogenesis. Which is in part due limitations in our current models of cardiogenesis, mainly relying on animal modeling or 2D in-vitro cultures, which lack the species-specific relevance, spatial organization and cellular crosstalk that is needed to fully understand HLHS development. Here, we utilize CRISPR/Cas9-mediated Notch1 knockout human induced pluripotent stem cells (hi-PSCs) as well as pharmacological inhibition in a hiPSC-derived cardiac organoid model to comprehensively evaluate the role of Notch signaling in HLHS pathology. Cardiac organoids were treated with Notch inhibitors DAPT or NADI-351 throughout the course of a 12-day cardiac differentiation. Organoids were imaged & collected throughout various developmental stages. DAPT & NADI-351 treated cardiac organoids exhibited decreased size and impaired function compared to control. We also utilized LC-MS proteomics & metabolomics of CRISPR-Cas9 Notch1 Knockout iPSC-derived cardiac organoids which revealed altered proteomic & metabolomic profiles throughout cardiac differentiation. Overall, these findings further highlight the advantage of cardiac organoid modeling for studying cardiogenesis and will allow us to gain a greater understanding of the complex interactions of Notch signaling and better understand its role in proper cardiac development.