Aim: To propose a new method to determine in vitro potentially bioavailable fluoride (F) in diet and toothpaste after ingestion. Methods: Diet samples (D) were obtained from 15 portions of a meal served to children in a day care centre. To simulate the ingestion of toothpaste during brushing after meals, a specific amount of toothpaste was added to the diet samples (D + T). F was determined in D and D + T after incubation in a solution that simulated “gastric juice” (0.01 M hydrochloric acid) at 37oC for 30, 60 and 120 min. Microdiffusion facilitated by HMDS was used to determine the total F concentrations in samples D and D + T. The analyses were performed using an ion specific electrode. Results: For D samples, incubation in “gastric juice” for 30, 60 and 120 min resulted in F concentrations (μg F/mL) of 0.75 ± 0.06c, 0.77 ± 0.07c and 0.91 ± 0.09b, corresponding to 75.3, 77.3 and 90.7% of the total F (1.02 ± 0.12a), respectively (p = 0.0001; ANOVA + Tukey). For D + T samples, these values of F concentrations (μg F/mL) were 2.55 ± 0.46b, 2.83 ± 0.44ab and 3.15 ± 0.37a, corresponding to 86.9, 94.8 and 106.7% of the total F (2.99 ± 0.34a), respectively (p = 0.0023; ANOVA + Tukey). Conclusion: Then, it can be concluded that the proposed method of “gastric juice” is a promising protocol for determining potentially bioavailable fluoride in the diet and toothpaste after ingestion. However, additional studies are desirable.