Purpose To determine the effect of proparacaine 0.375%-sodium fluorescein 0.25% eye drop mixture and fluorescein strip on anterior segment parameters commonly used in routine ophthalmology practice. Methods 115 healthy volunteers without any systemic or ocular disease were divided into two groups. 57 volunteers were in the proparacaine 0.375%-sodium fluorescein 0.25% eye drop mixture group, and 58 volunteers were in the fluorescein strip group. Measurements (CCT (central corneal thickness), topographic pupil diameter, AD (aqueous depth), ACV (anterior chamber volume), ICA (iridocorneal angle), LLD (limbus-limbus distance), and CV (corneal volume)) were taken before and at 1, 5, 15, and 30 minutes after application. Results 59 (51.3%) participants were female, and 56 (48.7%) were male. The mean age of the drop application group was 26.88 ± 8.03, and the mean age of the strip application group was 26.33 ± 7.28. The mean CCT was 556 ± 32 μm before drop application and 569 ± 30 μm in the 1st minute, 560 ± 32 μm in the 5th minute, and 559 ± 31 μm in the 15th minute. The change was statistically significant (p < 0.001, p=0.005, and p=0.013, resp.). Before the strip application, mean CCT was 552 ± 36 μm, while it increased to 556 ± 36 μm at the 1st minute, and this change was statistically significant (p=0.002). The mean CV before dropping was 59.29 ± 3.53 mm3 and 60.62 ± 3.53 mm3 at the 1st minute and 59.66 ± 3.70 mm3 at the 5th minute, which was statistically significant (p < 0.001 and p=0.034). Mean topographic pupil diameters at the 1st, 5th, 15th, and 30th minute after application of drops and strips were not significantly changed compared to the preapplication values in the AD, ACV, ICA, and LLD measurements. Conclusions Proparacaine 0.375%-sodium fluorescein 0.25% eye drop mixture and fluorescein strip application lead to a temporary change in CCT and CV of the anterior segment parameters. Anterior segment measurements with the Scheimpflug camera have to be done before topical fluorescein application.