OBJECTIVES Because of increases in antimicrobial resistance, the use of vancomycin in late-onset sepsis has come under scrutiny. The primary outcome of this study was to determine if vancomycin for the treatment of late-onset sepsis in the neonatal intensive care unit (NICU) was being discontinued within 72 hours according to the existing protocol. Secondary outcomes included the appropriateness of therapeutic drug monitoring associated with vancomycin, and renal dysfunction associated with the use of vancomycin in the NICU outside of the 72-hour policy. METHODS A retrospective chart review was completed for patients in the NICU who received vancomycin for the treatment of late-onset sepsis between the dates of January 1, 2014, and July 1, 2014. RESULTS There were 125 vancomycin treatment courses, of which 97 were included. Appropriate use of vancomycin, per policy, occurred in a total of 87 of 97 courses (89.6%). Therapeutic drug monitoring was evaluated by the number of appropriate troughs, determined using renal function and previous trough concentrations. There was not a statistically significant difference in the number of inappropriate troughs drawn between those that were continued on vancomycin appropriately (n = 17 courses; 4 of 44 inappropriate troughs) versus inappropriately (n = 10 courses; 1 of 22 inappropriate troughs; p = 0.66), despite the large number of troughs drawn. Adverse renal outcomes were not statistically significant in patients continued inappropriately on vancomycin (p = 1.0). CONCLUSIONS Vancomycin use in the NICU for late-onset sepsis is appropriate per the existing antibiotic policy. Therapeutic drug monitoring could be improved, and adverse renal outcomes due to inappropriate continuation of vancomycin are rare.