The respiratory activity of a multicellular spheroid was non-invasively monitored by scanning electrochemical microscopy (SECM) to investigate the anticancer drug sensitivity. The effects of the three anticancer drugs, cisplatin (CDDP), 5-fluorouracil (5-FU), and paclitaxel (TXL), were continuously evaluated based on respiratory activity for 5 days. The drug sensitivities obtained by SECM were higher compared to those evaluated by the spheroid volume and conformed to those evaluated by a conventional colorimetric assay. Our results show that the SECM-based assay directly correlates with the number of viable cells within the spheroid, whereas the spheroid volume does not necessarily correlate with the number of viable cells. Furthermore, the results obtained by spheroid culture (3-D) were compared to those of cells cultured in a flask (2-D) and within a collagen gel (3-D). The drug sensitivity of cells cultured in 2-D is more pronounced than that of the cells cultured in 3-D. Since the cellular proliferation status in a 3-D culture is similar to that in vivo, the drug sensitivity test performed in the spheroid culture will give meaningful results that can be extended to an in vivo application. A SECM-based assay is perfectly suitable to directly evaluate the drug sensitivity of the spheroid.