Quantitative investigation of the relationship between endosomal translation reactions and phospholipid membrane composition is crucial for enhancing protein translation efficiency in artificial cells. In this study, we quantitatively compared the translation reactions within liposomes containing negatively and positively charged lipids using green fluorescent protein fluorescence as an indicator to investigate whether lipid membrane charge affects translation reaction efficiency in artificial cells. Thus, translation efficiency reduced in liposomes containing both negatively and positively charged lipids. Interestingly, flow cytometry analysis revealed that the percentage of liposomes undergoing translational reactions was reduced by the charged phospholipids. This translation reaction inhibition was alleviated by adding equal amounts of negatively and positively charged lipids, indicating that phospholipid membrane charges affected translation reaction efficiency. The relationship between membrane composition and translation reaction efficiency identified in this study is significant for the constructing complex artificial cells, particularly concerning membrane composition design.