The food industry is moving away from synthetic colorants in favor of natural alternatives. Anthocyanins (ACNs) are plant pigments with vivid colors, but their application is restricted by their limited stability, particularly at pH close to neutral. Previous research showed that addition of whey protein isolate (WPI) to model juices colored with ACNs improved their color characteristics and increased their thermal resistance when compared with model juices without whey proteins (WP). It was hypothesized that WP may be interacting with ACNs, leading to enhancements in tinctorial strength and thermal stability. In this study, we performed fluorescence quenching (FQ) experiments to assess the level of interaction between anthocyanins and whey proteins. Eleven different anthocyanins were isolated from Berberis boliviana, purple corn and black carrot using semi-preparative HPLC. Identification and purity analyses were performed using uHPLC-MS. Model solutions were prepared by diluting WPI until a concentration of 1 mg/mL (0.1%, 54 μM) was reached using pH 3 citric acid-Na₂HPO₄ buffer, followed by the addition of purified ACNs (>90% purity) at different concentrations (1–100 μM of Cy-3-glu eq.). FQ by ACN addition was measured using a microplate reader with an excitation wavelength of 280nm. Results were adjusted to the Stern-Volmer (SV) equation for analysis. Furthermore, to stablish whether the FQ phenomena was a result of collisional quenching or binding-related quenching, measurements were conducted at different holding temperatures (30, 35 and 40°C). Results showed that addition of anthocyanins at concentrations as small as 1 μM resulted in significant FQ of WP (P < 0.05); and that Pt-3-glu, Mv-3-glu and Cy-3-glu were the most efficient quenchers. Measurements at different holding temperatures showed good linearity (r² > 0.9) and no significant changes in the SV constant with increasing temperature, suggesting that the type of FQ phenomena was a binding-related one. Therefore, WP can interact with anthocyanins in a binding-related manner, improving the color characteristics and thermal resistance of the pigment.