Osmoporation is an innovative method that can be used with food-grade yeast cells of Saccharomyces cerevisiae as natural encapsulating matrices. This technique overcomes barriers that difficult encapsulation and enables the internalization of fragile bioactive molecules such as fisetin into yeasts. In the present study, we assessed the effects of concentration, osmotic pressure, and temperature on the encapsulation efficiency (EE) and internalized fisetin content (IF). Two different quantification strategies were investigated: direct extraction (DE) without cell washing or freeze-drying steps and indirect extraction (IE) performed after washings with ethanol and freeze-drying. Our results showed that osmoporation improved EE (33 %) and IF (1.199 mg). The best experimental conditions were found by using DE. High-resolution images showed that the yeast cell envelope was preserved during osmoporation at 30 MPa and 84 % of yeast cells remained viable after treatment. Washing cells with organic solvent led to decreased EE (0.65 %) and IF (0.023 mg). This was probably due to either damages caused to yeast cell envelope or fisetin dragged out of cell. Overall, the results demonstrated the adequacy and relevant biotechnological potential of yeasts as encapsulating matrices for hydrophobic compounds. This fresh biotechnological approach has proven to be a promising tool for the production of bioactive-rich food products.