Brazil is one of the largest herd holders with a dependency on natural and cultivated pastures (Brachiaria species (100 million hectares)). The two most reported toxic pasture species (Brachiaria decumbens and Brachiaria brizantha) were grown in the greenhouse greenhouse of the Faculty of Animal Science and Food Engineering on different doses of nitrogen (0, 100, 200 and 300 mg/Kg) and were evaluated for a year. We explored the expected secondary metabolic compounds first by performing thin-layer chromatography, and then by confirming the compounds, their types and metabolic profiles, which were identified by ultra-high-performance liquid chromatography. Differential metabolites, steroidal saponin concentrations and their sub-types were confirmed by nano-magnetic resonance. The main source of differential probably toxic components in both species was the protodioscin isomer (Protoneodioscin and Protodioscin). We isolated four steroidal saponins and three sapogenins in leaves of Brachiaria decumbens and Brachiaria brizantha by MS-MS/MS-ESI/UPLC as diosgenin, dioscin, and yamogenin (already detected earlier) were detected. We identified protoneodioscin isomer (protodioscin and protoneodioscin (19 and 20) as the main differential (probably toxic)component between B. decumbens and B. brizantha along with acetyl-protodioscin, deoxyhexosyl-hexosyl-hexosyl-3-o-spirostane, and o-hexosyl-protodioscin in Brachiaria species. Additionally, the new findings included the fact that flavo-lignans, Tricin-4'-O-(β-guaiacyl glyceryl) ether isomers, acetyl-protodioscin, O-hexosyl-protodioscin, protodioscin isomer, tribulosaponin A and glycoside structures were also detected and established by ¹H and ¹³C NMR spectroscopy.