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Fourier transform infrared (FT-IR) spectroscopy is a physicochemical technique based on the vibrations of a molecule energized by infrared radiation at a specific wavelength range. Abiotic stresses can induce the production of secondary metabolites, increasing bioactivity. The objectives of the study were to evaluate the impact of heat treatments on the bioactivity of pineapple by-products, and whether FT-IR analysis allows understanding of the changes imparted by abiotic stress. The by-products were treated at 30, 40, and 50 °C for 15 min, followed by storage at 5 ± 1 °C for 8 and 24 h. Lyophilized samples were characterized for total phenolic content and antioxidant capacity and analyzed by FT-IR. Thermal treatments at 50 °C reduced the content of phenolic compounds (21–24%) and antioxidant capacity (20–55%). Longer storage time (24 h) was advantageous for the shell samples, although this effect was not demonstrated for the core samples. The principal components analysis (PCA) model developed with the spectra of the pineapple shell samples showed that the samples were grouped according to their total phenolic compounds content. These results allow the conclusion to be drawn that FT-IR spectroscopy is a promising alternative to the conventional chemical analytical methodologies for phenolic and antioxidant contents if there are significant differences among samples. [ABSTRACT FROM AUTHOR]