Introduction: Recently, natural dental bleaching agents are being developed to replace chemical dental bleaching agents. Chemical dental bleaching agents containing hydrogen peroxide have the potential to cause toxicity as a side effect. Spinach leaves are natural ingredients that contain oxalic acid which can whiten teeth. Purpose: To investigate the color changes in discolored human teeth after immersion in spinach extract in different durations. Methods: 30 human premolar samples were divided into 3 groups, group A (immersion in 28 hours), group B (immersion in 42 hours), and group C (immersion in 56 hours). All 30 samples were discolored with coffee solution for 12 days and color status (L₁, a₁, b₁) were measured using the chromameter. Then, each 10 samples were immersed in spinach extract according to their treatment group, and the color status (L₂, a₂, b₂) were measured again. Color changes (∆E) were calculated with a formula concerning L, a, b data measured. Results: A significant color change is observed between group A, group B, and group C (p-value= 0.002), and further comparison between three groups showed that group A differed significantly with group C (p-value=0.000), group B also showed significant difference with group C (p-value=0.038). However, there was no significant difference between group A and group B (p-value=0.121). Conclusion: The significant color changes can only be observed after 56 hours of immersion. The spinach leaf extract has the potential to be used as a natural dental bleaching agent.