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Prancak 95 tobacco has the potential to be developed in Indonesia because it has a distinctive aroma and a nicotine content of 2.13%. The quality of this tobacco raw material must be improved and started from the use of quality seeds through in vitro culture techniques. However, several things that must be considered are the acclimatization stages. One of the efforts to increase the growth of seedlings during the acclimation can be done by providing natural plant growth regulators (PGR) from extracts of organic materials such as coconut water, young corn, sprouts, and banana weevil that has been fermented in advance using EM-4 bioactivators . Based on this, the purpose of this study was to determine the optimal concentration of natural growth regulators in the acclimatization of Prancak -95 tobacco. This study uses a non factorial randomized block design with four treatments concentrated solution of natural growth regulators, which include: Control (no treatment), 10; 25; and 50 ml/L. The observed variables included the number of leaves, leaf area, plant height, and root length. Data analysis using ANOVA Test and continued with DMRT Test at 95% confidence level to find out the best treatment. Based on the results of research that has been carried out for 60 days of observation, it can be concluded that the application of natural PGR is able to spur the growth of seedlings during the acclimatization period. Spraying application at a concentration of 10 ml / L can increase the number of leaves, leaf area, number of roots and height of Prancak 95 tobacco seedlings.