Preference for a pupation site is a crucial phase in Drosophila's pre-adult behavioral development since the location a larva chooses may significantly affect its chances of surviving as a pupa. In this study, PSP was investigated using D. melanogaster larvae cultivated in wheat-cream agar and combined (wheat-cream agar + caffeine powder) media. Pupae in wheat cream agar media tended to pupate more frequently in the following order: on the media's surface, on its wall, and next to the cotton plug. However, the larvae cultivated in the test (caffeine) media prefer to pupate more frequently on the media bottle wall, then close to the cotton plug, and less frequently on the media surface. In contrast, the PSP varies depending on the concentration of caffeine media (2.5g, 5g, and 10g). In 2.5g, the larvae prefer to pupate primarily on the media bottle wall, followed by the media surface and less on the cotton plug; in 5g, the larvae prefer to pupate primarily on the media bottle wall, followed by the cotton plug and less on the media surface; and in 10g, the larvae prefer to pupate primarily on the media bottle wall, followed by the cotton plug and very little on the media surface.