Antimicrobial use (AU) in days of therapy per 1000 patient-days (DOT/1000 pd) varies widely among children's hospitals. We evaluated indirect standardization to adjust AU for case mix, a source of variation inadequately addressed by current measurements. Hospitalizations from the Pediatric Health Information System were grouped into 85 clinical strata. Observed to expected (O:E) ratios were calculated by indirect standardization and compared with DOT/1000 pd. Outliers were defined by O:E z-scores. Antibacterial DOT/1000 pd ranged from 345 to 776 (2.2-fold variation; interquartile range [IQR] 552-679), whereas O:E ratios ranged from 0.8 to 1.14 (1.4-fold variation; IQR 0.93-1.05). O:E ratios were moderately correlated with DOT/1000 pd (correlation estimate 0.44; 95% confidence interval, 0.19-0.64; P = .0009). Using indirect standardization to adjust for case mix reduces apparent AU variation and may enhance stewardship efforts by providing adjusted comparisons to inform interventions.