Biduri (Calotropis gigantea) is a plant that has a great opportunity to be used as a raw material for textile fibers. However, biduri has not been widely cultivated and many people still consider biduri as a weed. In vitro cultivation of biduri can be done for the propagation of biduri and produce superior and uniform seedlings. Factors that affect the cultivation of tissue culture include the growing media and plant growth regulator, environmental factors around the cultivation area, and the type of explant used. Plant Growth Regulator (PGR) used are NAA from the auxin group and BA from the cytokinin group. NAA is used with the aim of stimulating root formation whereas BA aims to stimulate the formation of buds. The research was conducted in August-December 2021 and was carried out at the Laboratory of Plant Physiology and Biotechnology of the Faculty of Agriculture, Sebelas Maret University, Surakarta. The design used is Completely Randomized Design with 2 factors. The first factor was the concentration of NAA consisting of 5 levels including 0; 0,5; 1; 1,5; and 2 ppm. The second factor was the concentration of BA consisting of 5 levels including 0, 1, 2, 3, and 4 ppm. The data was analyzed using the F test with a level of 5%. If the test results show a significant effect, further tests are carried out using the 5% Duncan Multiple Range Test. The variables measured in the study were the time of budding, the time of root appearing, the time of callus appearing, the color of callus, the texture of callus, the number of shoots, the height of the shoots, the number of leaves, the number of roots, and the length of the roots. The best planlet growth response was found in media combined with concentrations of 0 ppm NAA and 4 ppm BA. This was indicated by the large number of shoots formed, optimal shoot height, and no callus formation on planlet. Increasing the concentration of NAA can slow down root formation and reduce the number of leaves and root length, while high and low concentrations of NAA can induce callus formation. The administration of single ZPT BA did not have a significant effect on the growth of biduri plants.