Probiotics play important roles in many crucial functions for maintaining the homeostasis of the host, such as protection against pathogens, immunity and metabolism of dietary compounds. Resistant starch (RS) is the starch or the starch products which cannot be digested through the body, but can be digested by the gut microbiota, producing a variety of metabolites which can provide a range of physiological benefits to the host. An RS can be classified as a prebiotic if it can selectively stimulate the growth and/or activity of the beneficial bacteria. To investigate the bacteria driven structural changes in the prebiotic tapioca starch, we used Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopy. Principal Component Analysis (PCA) and intensity analyses of individual spectral bands exhibited comprehensive alterations in the polysaccharide composition of the tapioca starch incubated with the probiotic bacteria; the starch samples incubated with the bacteria gained a more amorphous structure with a decrease in the ordered structure. The results suggest that as a fast, cheap, and non-laborious method, FTIR spectroscopy coupled with PCA has a potential to be applied in the research area as well as in the food industry for the analysis of the potential prebiotic activity of starch-based substrates or the investigation of the probiotic potential of a bacterial strain through the examination of its RS hydrolyzing capacity.