This case report describes a novel mutation of the SPTB gene as a potential pathogenic cause of spherocytosis. A three-week-old male presented with clinical and laboratory signs consistent with hemolytic spherocytosis (HS), including jaundice, hyperbilirubinemia, anemia, reticulocytosis, negative Coombs test, no ABO or Rh incompatibility, and a peripheral blood smear notable for numerous spherocytes. His lab work demonstrated persistent anemia despite daily folate prompting next-generation sequencing (NGS) which revealed a novel mutation in the SPTB gene resulting in a non-functioning protein product. Correlation of the genetic finding with clinical presentation may help guide management for this and future patients.