The objective of this study was to determine and compare the cost to treat HIV(+) and HIV(-) pediatric patients both before and after HIV prophylaxis became the standard of care. Retrospective chart review of a pediatric HIV/AIDS specialty clinic's medical charts was conducted for clinical and healthcare utilization data on 125 children diagnosed from 1986 to 2007. Mean HIV-related costs were compared using bootstrapped t-tests for children born in the pre-prophylaxis (1979-1993) and prophylaxis eras (1994-2007). Patients were also stratified into two categories based on death during the follow-up period. Lastly, national cost-savings were estimated using mean costs, national number of at-risk births, and national perinatal HIV transmission rates in each era. For HIV(+) children, mean annual per patient treatment cost was $15,067 (95% CI: $10,169-$19,965) in the pre-prophylaxis era (n = 40) and $14,959 (95% CI: $9140-$20,779) in the prophylaxis era (n = 14); difference not statistically significant (p > 0.05). For HIV(-) children, mean annual per patient treatment cost was $204 (95% CI: $219-$627) for the pre-prophylaxis era (n = 2) and $427 (95% CI: $277-$579) for the prophylaxis era (n = 69); difference statistically significant (p < 0.05). A projected cost-savings of $16-23 million annually in the USA was observed due to the adoption of prophylaxis treatment guidelines in pediatric HIV care. The prophylaxis era of pediatric HIV treatment has been successful in decreasing perinatal HIV transmission and mortality, as reflected by clinical trials and national cost-savings data, and emphasizes the value of the rapid adoption of evidence-based practice guidelines.