Objective: To compare costs between two different eye drop delivery modalities: multidose bottles and single-use minims. Design: Retrospective cohort study. Methods: Monthly dilating eye drop costs and quantities (tropicamide 1%, phenylephrine 2.5%, cyclopentolate 1%) were studied over a 2-year period between April 2013 and March 2015 at 2 tertiary ophthalmic centres (Royal Alexandra Hospital [RAH, Edmonton] and Rockyview General Hospital [RGH, Calgary]). In April 2014, RAH switched its dilating eye drop practice from predominantly multidose bottles to single-use minims, whereas RGH continued using predominantly multidose bottles. Eye drop volume and total and per-patient eye drop costs were quantified at RAH before switching (pre-intervention) and after (post-intervention) using an interrupted time-series analysis with RGH as a control. A counterfactual analysis was also performed. Significance was obtained using independent t-testing. Results: After switching to single-use minims, RAH experienced changes in the following: an increase in single-use minims as a proportion of total eye drop utilization (from 5.6% to 89.1%; p = 0.001), an increase in total eye drop cost by $2117 per month (95% confidence interval [CI], $1354–$2880; p < 0.001), an increase in per-patient costs by $984 per 1000 patients per month (95% CI, $674–$1293). Contrastingly, RGH did not experience similar changes. Ultimately, the cost of switching to single-use minims was $22 481 (95% CI, $7830–$31 336) over a 12-month period. Conclusions: If safe eye drop practices are enforced via proper protocols, the use of multidose bottles may be a more cost-effective option than single-use minims for routine clinical practice.