A derivatisation-free HPLC–MS/MS method was developed and validated for the simultaneous quantification of selenium- and sulphur-containing amino acids in soybean leaves, and applied to a 3 × 3 factorial hydroponic experiment probing selenium–sulphur metabolic interactions. The method resolves five biologically informative analytes (Cys₂, SeCys₂, MeSeCys, Met, SeMet) within 1.5 min through multiple reaction monitoring (MRM). Ultrasound-assisted extraction (UAE) of the free fraction was jointly optimised for both analyte classes by the response-surface methodology; enzymatic hydrolysis of the extraction residue recovered the protein-bound fraction on the same platform. Limits of detection ranged from 0.036 to 0.556 µg L⁻¹, intra-day relative standard deviations were below 5%, and spike recoveries fell between 92.3 and 117.4%. Free SeAA and SAA pools were negatively correlated across the nine treatments (R² = 0.83), consistent with competitive Se–S assimilation, whereas bound pools were positively correlated (R² = 0.89), reflecting proportional protein-level incorporation. A regime of 1–5 mM of sulphate with 20 µM of selenite yielded the highest bound organo-Se with near-normal growth, providing leaf-level evidence that may inform future seed-focused studies aimed at Se-enriched soy-protein ingredient development.