Introduction: The right ventricular outflow tract (RVOT) is pathologically affected in multiple congenital heart diseases, requiring surgical replacement within the first year of life. Shortage of small-sized homografts has driven xenograft use in pediatric cardiac surgery. However, these RVOT substitutes do not possess lifelong durability, eliciting high levels of inflammation and lacking growth potential. Xenograft limitations culminate in structural graft deterioration and rejection, resulting in invasive reoperations. Decellularization of donor tissue to eliminate cells and immunogenic antigens is a central method by which rejection risk can be diminished. Maintenance of the extracellular matrix (ECM) must be balanced with achievement of acellularity. Currently, no xenograft has proven optimal for pediatric RVOT surgical replacement; this work aims to produce a resilient and non-immunogenic decellularized porcine scaffold with translational capacity. Methods/Results: Research sought to optimize a combined chemical, enzymatic, and mechanical decellularization approach for RVOT conduits. The nine-day protocol utilizes a temperature-controlled bioreactor wherein EDTA, protease inhibitors, sodium dodecyl sulfate, and nucleases are circulated through the lumen of the submerged conduit. Maintenance of ECM structure, including elastin and collagen, in parallel with significant nuclei count reduction, removal of residual nucleic acids, and elimination of xenoantigens has been achieved. No significant difference was found between control and decellularized ventricular soluble collagen. All comparisons between samples processed directly after explant (fresh) and those decellularized following one freeze-thaw cycle (freeze-thawed) were not significantly different. Conclusion: Data indicate successful decellularization of valved conduits. Future work intends to seed mesenchymal stem cells onto the cell-free scaffold, improving biocompatibility and endowing the xenograft with growth potential. Decellularized RVOT xenografts provide a means to combat immunological mismatch between donor and patient, reducing the number of surgeries a child is exposed to and improving pediatric congenital surgery outcome.