It has been proposed that the composition of the culture medium, especially its amino acids, is an important part of getting microspore androgenesis to occur in some plants. However, there have been far fewer studies done on the Solanaceae family. In this study, we studied what happened to eggplant microspore culture when we mixed casein hydrolysate (0 and 100 mg L-1) with four amino acids: proline (0, 100, 500, and 900 mg L-1), glutamine (0 and 800 mg L-1), serine (0 and 100 mg L-1), and alanine (0 and 100 mg L-1). The results showed that a combination of 800 mg L-1of glutamine, 100 mg L-1of serine, 100 mg L-1of casein hydrolysate, and 500 mg L-1of proline produced the maximum number of calli per Petri dish (938). Calli had a globular shape and a compact appearance when formed in media containing 500 mg L-1of proline (alone or combined with serine, alanine, and/or casein hydrolysate). Most of these structures were observed in a medium with 500 mg L-1of proline, 100 mg L-1of casein hydrolysate, and 100 mg L-1of serine. We also investigated what happened when gum arabic (2400, 2600, 3600, 4600, and 5600 mg L-1) was combined with proline (0 and 500 mg L-1), casein hydrolysate (0 and 100 mg L-1), and glutamine (0, 400, and 800 mg L-1). The findings demonstrated the involvement of proline in the increase of calli. Overall, the results give us new information about how amino acids work in eggplant microspore culture and suggest that proline can move this plant’s microspore androgenesis pathway forward.