Globe artichoke’s [Cynara cardunculus var. scolymus (L.) Fiori] leaves are rich in polyphenols and due to health-promoting properties artichoke growing has been gaining interest. Optimization and development of valuable bioactive components, which are not in the standard amount in raw material can be achieved and increased with the assistance of in vitro techniques such as callus and subsequently cell suspension cultures. Therefore, in the present study in vitro callogenesis optimization of three globe artichoke cultivars was studied by using 29 different media combinations, based on basic Gamborg B5 medium supplemented with various concentrations of 1-Naphthaleneacetic acid (NAA), 6-Benzylaminopurine (BAP), 2,4-Dichlorophenoxyacetic acid (2,4-D), and Kinetin. Comparisons were made on the basis of using in vivo and in vitro leaves as explant material. In the experiment several parameters such as leaf explants development (%), callus formation (%), and callus weight (g) were assessed for each related cultivar. Results revealed that having auxin: cytokinin concentrations together at enough and well-balanced, having equal amounts or 10:1 concentrations of auxin: cytokinin, concentrations in media combinations are indispensable for stimulating the callogenesis in globe artichoke. The findings of the present study clearly revealed that, there were differences among cultivars regarding callus induction by using in vivo and in vitro leaf explants while in vivo leaf explants came into prominence regarding callus formation and weights. It is assumed that the findings of the present study may play a complementary and auxiliary role in several areas such as pharmaceutical engineering of globe artichoke.