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The food industry is increasingly innovating and applying new processing technologies and ingredients to develop novel food products that meet the consumers’ demand. In this study, the effect of extrusion (at 140 °C and 160 °C) was evaluated in different lentil flours formulations enriched with nutritional yeast, in terms of α-galactosides (raffinose, stachyose, verbascose), inositol phosphates (IPs), trypsin inhibitors and lectins content. The content of α-galactosides and IPs was determined by high performance liquid chromatography. Trypsin inhibitor activity (TIA) was evaluated using a small-scale quantitative assay. The lectin content was analyzed using a haemagglutination assay and a Competitive Indirect Enzyme-linked immunosorbent assay. Extrusion promoted a significant increase, up to 85% in total α-galactosides content. After extrusion, IPs content was significantly decreased and TIA as well as lectins …