Microphysiological systems have attracted attention because of their use in drug screening. However, it is challenging to measure cell functions in real time using a device. In this study, we developed a cell culture device using a porous membrane electrode for in situ electrochemical glucose measurements for cell analysis. First, a porous membrane electrode was fabricated and electrochemically evaluated for enzyme-free glucose measurement. Subsequently, the glucose uptake of MCF-7 spheroids was evaluated using living spheroids, fixed spheroids, supernatants, and glucose transporter inhibitor-treated spheroids. Conventionally, the direct optical measurement of glucose uptake requires fluorescence-labeled glucose derivatives. In addition, the glucose uptake can be evaluated by measuring the glucose concentration in the medium by optical or electrochemical measurements. However, glucose needs to be consumed in the entire cell culture medium, which needs a long culture time. In contrast, our system can measure glucose in approximately 5 min without any labels because of in situ electrochemical measurements. This system can be used for in situ measurements in in vitro cell culture systems, including organ-on-a-chip for drug screening.