Purpose: The purpose of this study is to investigate and compare the effects of cyclopentolate and tropicamide drops on anterior segment parameters in healthy individuals. Methods: Two hundred and fifty-eight eyes of 129 healthy volunteers were included in this randomized clinical study. Cyclopentolate 1% drop was applied to 75 (58%) participants (group 1) and tropicamide 0.5% drop was applied to 54 (42%) participants (group 2). Flat keratometry (K1), steep keratometry (K2), axial length (AL), central corneal thickness (CCT), anterior chamber depth (ACD), white-to-white (WTW) distance, pupil diameter, total pupil offset and intraocular lens (IOL) power were measured before and after drops, using Lenstar 900 optical biometry. Results: The increase in CCT, ACD, pupil diameter, and pupil offset was significant in group 1 after the drop (P < 0.05), while the increase in ACD, pupil diameter, and pupil offset was significant in group 2 (P < 0.05). When the two groups were compared, there was no significant difference in K1, K2, CCT, ACD, WTW, pupil diameter, pupil offset, and IOL power (using Sanders-Retzlaff-Kraff T formula) changes after drops (P > 0.05), whereas the change in AL was significant (P = 0.01). Conclusion: The effects of cyclopentolate and tropicamide drops on anterior segment parameters were similar; they did not make significant changes in K1, K2, AL, WTW, and third-generation IOL power calculation. However, ACD values significantly changed after these drops; thus, measuring anterior segment parameters before mydriatic agents should be taken into account particularly for fourth-generation IOL formulas and phakic IOL implantation. The change in pupil offset, which can be important in excimer laser and multifocal IOL applications, was not clinically significant.