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Monitoring of metabolic compounds in cell cultures can provide real-time information of cell line status. This is particularly important in those lines not fully known, as the case of embryonic and mesenchymal cells. On the other hand, such approach can pave the way to fully automated systems for growing cell cultures, when integrated in Petri dishes. To date, the main efforts emphasize the monitoring of few process variables, like pH, pO2, electronic impedance, and temperature in bioreactors. Among different presented strategies to develop biosensors, carbon nanotubes exhibit great properties, particularly suitable for high-sensitive detection. In this work, nanostructured electrodes by using multiwalled carbon nanotubes are presented for the detection of lactate and glucose. Some results from simulations are illustrated in order to foresee the behavior of carbon nanotubes depending on their orientation, when they are randomly dispersed onto the electrode surface. A comparison between nonnanostructured and nanostructured electrodes is considered, showing that direct electron-transfer between the protein and the electrode is not possible without nanostructuration. Such developed biosensors are characterized in terms of sensitivity and detection limit, and are compared to previously published results. Lactate production is monitored in a cell culture by using the developed biosensor, and glucose detection is also performed to validate lactate behavior.