The heart is the first major organ to develop during embryogenesis and must receive proper spatiotemporal signaling for proper development. Failure of proper signaling between the first and second heart fields at twenty days gestation contributes to the generation of a congenital heart defect. The most common cyanotic congenital heart defect is tetralogy of Fallot (TOF) which requires surgical intervention in the first year of life. In right ventricular tissue of infants born with TOF, the levels of scaRNA1 are reduced and mRNA splicing is dysregulated. In this study, we investigate a method of quantifying pseudouridylation levels in relation to scaRNA1 levels in spliceosomal RNA U2 in three different groups of samples: right ventricular (RV) tissue of infants born with TOF versus RV tissue from normally developing infants, scaRNA1 knockdown in primary normal cardiomyocytes derived from normally developing infants, and scaRNA1 overexpression in primary cells derived from RV tissue from infants born with TOF. We hypothesize that the amount of pseudouridylation is dependent on scaRNA1 level, compromising spliceosomal function and therefore, contributing to the generation of a congenital heart defect. Our results revealed a statistically significant decrease of pseudouridylation levels in the right ventricular tissue of infants born with TOF compared to the controls. Knocking down the scaRNA1 levels in normal primary cardiomyocytes resulted in a statistically significant decrease of pseudouridylation. Finally, an overexpression of scaRNA1 in TOF primary cells resulted in an increase in pseudouridylation levels, but it did not achieve statistical significance. Our previous research provided an association between scaRNA levels, alternative splicing, and development. Here, we demonstrate that pseudouridylation levels in spliceosomal RNA U2 is dependent on the expression level of scaRNA1. Although further investigation is needed, we believe that scaRNA expression regulates biochemical modifications to spliceosomal RNAs, adjusting the fidelity of the spliceosome, allowing for controlled alternative splicing of mRNA that is important in embryonic development. If validated, this is an underappreciated mechanism that is critical for regulating proper embryonic development.