The Cerrado is a biome in Brazil that is experiencing the most rapid loss in natural vegetation. The objective of this study was to analyze the changes in the spectral response in the red, near infrared (NIR), middle infrared (MIR), and normalized difference vegetation index (NDVI) when native vegetation in the Cerrado is deforested. The test sites were regions of the Cerrado located in the states of Bahia, Minas Gerais, and Mato Grosso. For each region, a pair of Landsat Thematic Mapper (TM) scenes from 2008 (before deforestation) and 2009 (after deforestation) was compared. A set of 1,380 samples of deforested polygons and an equal number of samples of native vegetation have their spectral properties statistically analyzed. The accuracy of deforestation detections was also evaluated using high spatial resolution imagery. Results showed that the spectral data of deforested areas and their corresponding native vegetation were statistically different. The red band showed the highest difference between the reflectance data from deforested areas and native vegetation, while the NIR band showed the lowest difference. A consistent pattern of spectral change when native vegetation in the Cerrado is deforested was identified regardless of the location in the biome. The overall accuracy of deforestation detections was 97.75%. Considering both the marked pattern of spectral changes and the high deforestation detection accuracy, this study suggests that deforestation in Cerrado can be accurately monitored, but a strong seasonal and spatial variability of spectral changes might be expected.