Newborn screening (NBS) for Cystic Fibrosis (CF) has revolutionized the diagnosis of this inherited disease. CF NBS goals are to identify, diagnose, and initiate early CF treatment to attain better health outcomes. Abnormal CF NBS infants require diagnostic analysis via sweat chloride testing (ST). During ST, insufficient sweat volume collection causes a “quantity not sufficient” (QNS) test result and may delay CF diagnosis. The CF Foundation recommends QNS rates <10% for infants <3 months, but many CF Centers experience difficulties meeting this standard. Our quality improvement (QI) study assessed infant and laboratory factors contributing to ST success and QNS rates from 2017–2019. Infants’ day of life (DOL) at successful ST completion was analyzed according to infant factors (birth weight (BW), gestational age, ethnicity, and sex). Laboratory factors and procedures affecting ST outcomes were also reviewed. At our institution, BW and gestational age were the infant factors found to significantly affect DOL at ST completion. ST education, reduced number of laboratory technicians, and direct observation during ST completion also improved ST success rates. This study supports QI measures and partnerships between CF centers and laboratory staff to identify and improve ST QNS rates while sustaining practices to ensure timely CF diagnostic testing.