This study was conducted to determine the effects of different culture times on root growth and accumulation of secondary metabolites in adventitious roots from Hyoscyamus niger petioles. For this purpose, adventitious roots transferred to culture media containing 1 mM methyl jasmonate (MeJA) were harvested at 11 different culture times, starting from day 1 to day 30. After harvest, the fresh and dry weights of the roots and the root growth index as parameters of root growth, as well as the amounts of tropane alkaloids and phenolic compounds, were examined. It was found that the fresh and dry weights and the growth index of the roots harvested between the 12th and 30th day were the highest. The highest amounts of scopolamine were obtained from roots harvested on days 15, 18, and 21. The highest hyoscyamine accumulation was found on days 12 and 15. The highest total phenolic content was 19.33 mg g-1 in root cultures harvested on day 15. HPLC analyses revealed that ferulic acid, caffeic acid, o-coumaric acid, p-coumaric acid, rutin, and quercetin were not detected in the roots. On the other hand, the amounts of gallic acid, catechin, epicatechin, vanillin, cinnamic acid, rosmarinic acid, and chlorogenic acid showed significant changes depending on the culture times. Considering all the results, it was found that the optimum culture time for the production of secondary metabolites in H. niger adventitious root cultures stimulated with 1mM MeJA was 15 days.