Background: The aim of this study was to compare osmotic pressure (OP) 1 week after the gingival depigmentation in two diffeent methods: Er: YAG laser and Kirkland knife. Methods: This study included 20 patients, 13 women, and 7 men who had esthetic complaints of gingival melanin hyperpigmentation. The left side of the maxilla and mandible were depigmented with Er:YAG laser using air and water spray and the right side was removed with a Kirkland knife. Gingival biopsies were obtained from attached gingiva of operation sides with approximate dimensions of 2 mm × 2 mm before and 1 week after the depigmentation. The visual analog scale (VAS) was used to evaluate the pain level experienced by each patient. Results: Patients reported no pain at the gingivoplasty sides after 4 days and at the laser sides after 3 days. There were not statistical differences between laser and gingivoplasty groups according to values of OP (P > 0.05). Conclusion: According to results of wound healing, OP and VAS, two depigmentation procedures can be used safely and effectively.