OBJECTIVE To determine whether the mydriatic efficacy of spray application of tropicamide 1% is comparable to drop instillation of tropicamide 1%, and to compare the ocular discomfort caused by these methods. METHODS Thirty-four healthy volunteers were randomly assigned to one of two groups, and received either a single drop of tropicamide 1% eye drops or a single puff of tropicamide 1% spray into open eyes. Pupil diameters were measured from anterior segment images taken using a Topcon Imagenet system at baseline and at the fifth, tenth and fifteenth minute after drug administration. Ocular discomfort experienced with each method was also compared. RESULTS Repeated measures analysis of variance revealed that a statistically significant increase in pupil diameter was achieved with both application methods over time (p < 0.0001), and that there were no statistically significant differences in pupil diameter between the two groups at each time point (p = 0.409). The mean ocular discomfort score for tropicamide 1% spray was 1.45 +/- 0.56, and for tropicamide 1% eye drops was 2.71 +/- 0.67. This difference was statistically significant (p < 0.001). CONCLUSIONS The mydriatic efficacy of tropicamide 1% spray is similar to that of conventional tropicamide 1% eye drops, and spray application causes less ocular discomfort.