Potential impurities in a parenteral infusion solution for amino acid supplementation containing alanylglutamine (AlaGln) and glycyltyrosine (GlyTyr) as peptide constituents have been determined. Such complex multicomponent pharmaceutical formulations with reactive ingredients may yield a multitude of impurities in stress testing samples. Thus, three stability indicating LC–ESI-MS/MS methods were developed for the establishment of quantitative impurity profiles employing a Chiralpak QN-AX and a Polysulfoethyl A stationary phase in HILIC mode as well as a Gemini C18 stationary phase in gradient RPLC mode. The primary goal was to separate isobaric compounds (stereoisomers, constitutional isomers, retro-peptides) and to provide quantitative data of impurities identified in stressed nutritional infusion solutions. The optimized methods were calibrated by standard addition in the samples and validated according to ICH guidelines. The methods were then applied for the analysis of stressed sample solutions stored under different conditions. Major peptide impurities found in concentrations above the qualification threshold in stressed solutions stored at 40°C for 6 months comprised cyclo(AlaGln) 808μg/mL, pyroGluAla 122μg/mL, AlaGlu 117μg/mL, cycloGlyTyr 60μg/mL, AlaGln epimers (DL+LD) 38μg/mL, and TyrGly 27μg/mL. A number of impurities above the reporting threshold were also detected including AlaAlaGln 18μg/mL, cyclo(AlaGlu) 16μg/mL, AlaGlu(AlaGln) 17μg/mL, and AlaGlu(His) 12μg/mL. The study showed that bioactive peptides may be formed in amino acid infusion solutions by condensation of amino acids and a careful control of these impurities is mandatory.