Fetal bovine serum (FBS) has been an essential element in long-term in vitro cell culture experiments due to it containing diverse nutrients for cell viability. However, recent movements to reduce livestock cultivation and frequent outbreaks of infectious diseases in cattle have posed challenges in the continuous production of FBS. Moreover, given the increased development of pharmaceuticals utilizing cell cultures, the price of FBS has risen. To address this issue, we developed a medium that only requires half the amount of FBS when culturing cells. We named this medium Half-SerumTM DMEM, and we demonstrate here that during a 20-days culture, its performance aligns with that of the existing medium in terms of cell cycle, morphology, isoform protein expression, lentivirus production, cell growth, cell death, and the regulation of inflammation through major signaling pathways, even after cell culturing. Half-SerumTM DMEM will play a pivotal role in alleviating the financial burden on medical scientists. This will ultimately contribute to cost savings regarding patients' healthcare expenses. Finally, these advancements are expected to have a positive impact on both economic and environmental aspects.