The present study has investigated the effect of oil rich medium supplementation with polysorbates Tween 20, 40 and 80, for the cultivation of red oleaginous yeast Rhodotorula glutinis. R. glutinis has been cultivated in yeast extract peptone glucose modified broth (mYPG) supplemented with 2 % of waste cooking rapeseed oil and three polysorbate types with 0.5 %, 1 %, 2%, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 % and 10 % concentration each. Yeast biomass was measured by the thermogravimetric method at 105 °C each day during 7-day experiment. The oil rich medium supplementation with Tween 20, 40 and 80 at concentrations ranging from 2 % to 10 % significantly increased the biomass of R. glutinis. All three types of the studied polysorbates with 0.5 % and 1 % concentration, did not affect yeast growth and the dry biomass – results were similar to the control sample without polysorbate addition. Between the three types of polysorbates, Tween 20 was selected as the preferable for R. glutinis cultivation with an optimal concentration of 2 %. Cultivation of R. glutinis in oil rich medium with polysorbates Tween 20, 40 and 80, supplementation up to 10 % concentration did not have had an inhibitory effect on the biomass growth.