Background: Antibiotics are widely used in very low-birth-weight infants (VLBW, <1500 g), and excess exposure, particularly to broad-spectrum antibiotics, is associated with significant morbidity. An antibiotic spectrum index (ASI) quantifies antibiotic exposure by relative antimicrobial activity, adding information to exposure measured by days of therapy (DOT). We compared ASI and DOT across multiple centers to evaluate differences in antibiotic exposures. Methods: We extracted data from patients admitted to 3 level-4 NICUs for 2 years at 2 sites and for 1 year at a third site. We calculated the ASI per antibiotic days and DOT per patient days for all admitted VLBW infants <32 weeks gestational age. Clinical variables were compared as percentages or as days per 1,000 patient days. We used Kruskal-Wallis tests to compare continuous variables across the 3 sites. Results: Demographics were similar for the 734 VLBW infants included. The site with the highest DOT per patient days had the lowest ASI per antibiotic days and the site with the highest mortality and infection rates had the highest ASI per antibiotic days. Antibiotic utilization varied by center, particularly for choice of broad-spectrum coverage, although the organisms causing infection were similar. Conclusion: An antibiotic spectrum index identified differences in prescribing practice patterns among 3 NICUs unique from those identified by standard antibiotic use metrics. Site differences in infection rates and unit practices or guidelines for prescribing antibiotics were reflected in the ASI. This comparison uncovered opportunities to improve antibiotic stewardship and demonstrates the utility of this metric for comparing antibiotic exposures among NICU populations.