Background: Calcium oxalate (CaOx) stones are the most prevalent type of urolithiasis and are associated with high recurrence rates. Preventive approaches targeting early crystallization stages, particularly nucleation and aggregation, are therefore crucial. Citrate is a recognized inhibitor of CaOx crystallization; however, pharmacological citrate therapy may face limitations in long-term use. Lime juice (Citrus aurantifolia), a natural source of citrate, may serve as an alternative preventive agent. Objective: To evaluate the inhibitory effect of lime juice (Citrus aurantifolia) on calcium oxalate crystal formation in vitro. Methods: A true experimental study with a post-test only control group design was conducted using a synthetic urine model. Five groups were assessed: negative control, positive control (potassium citrate 0.6%), and lime juice at concentrations of 2.5%, 5%, and 10% (v/v). Crystallization was monitored turbidimetrically at 620 nm for 60 minutes. Nucleation slope (SN), aggregation slope (SA), time to maximum absorbance (Tmax), and inhibition percentages were analyzed using one-way ANOVA and Tukey’s HSD test. Results: All lime juice concentrations significantly reduced aggregation and prolonged Tmax compared with the negative control. The 5% concentration showed the strongest nucleation inhibition among natural treatments, while 2.5% demonstrated the highest aggregation inhibition. Conclusion: Lime juice effectively inhibits CaOx crystallization in vitro and shows potential as a natural, accessible preventive strategy.