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Cupuassu (Theobroma grandiflorum Schum.) is a typical Amazonian fruit, whose seed is used as raw material to produce cupulate. The by-product of its seeds, cupuassu seed by-product (CSB), is often discarded by the cosmetic and food industry, but studies showed that it has a high content of phenolic compounds with exploitable antioxidant activity. This study aimed to identify the classes of chemical compounds present in the extract of CSB by HPTLC, to confirm them by RMN 1H spectroscopy and to analyze the extract thermal analytical behavior in different processing stages. After elution of the extract in an isocratic system, TLC plates were developed with selective reagents for several classes of chemical compounds and antioxidant activity by the DPPH· method. Butter, CSB and crude extract presented mass losses characteristic of lipid decomposition reactions (92.63, 14.80, and 15.42%, respectively). Thermal degradation of bioactive and nutritional compounds (mainly polyunsaturated fatty acids) present in both CSB and crude extract has occurred at temperatures above 189 °C. Microencapsulation of the CBS extract by spray drying (MECoC) using maltodextrin as coating agent was shown to effectively protect the phenolic compounds. RMN 1H spectra confirmed HPTLC and FTIR results, which allowed us to conclude that the crude extract of the cupuassu by-product was mainly composed of terpenes, steroids and phenolic compounds, mainly flavonoids. These analytical techniques have proven to be effective tools for assessing the bioactive compounds incorporation into the polymeric matrix expected in the pre-formulation stage. [ABSTRACT FROM AUTHOR]