The detection of the herbicide glyphosate (GLP) in environmental samples is most often conducted after derivatizing the target molecule with the chromophore 9-fluorenylmethyloxycarbonyl chloride (FMOC-Cl). However, this method is sensitive to all primary and secondary amines, which can occur in the sample matrix as well. In order to quantify the interference of primary and secondary amines on GLP detection, we have used well-defined peptides such as pentaglycine (PG) and albumin as well as mixtures of peptides such as peptone. These peptides have been added to the derivatization solution of GLP at different constant concentration levels and UV extinction coefficients have been determined. Data analysis supported by quantum chemical modeling of the GLP–peptide, FMOC–GLP, and FMOC–peptide complexation reactions facilitated the identification of two interfering impacts of peptide on GLP derivatization: (i) increase of the signal due to reaction with FMOC-Cl leading to an overestimation of GLP concentration and (ii) decrease of GLP recovery due to complex formation and therefore inhibition of GLP derivatization, which leads to an underestimation. Specifically, our results indicated that the GLP-peptide- and peptide-FMOC-interactions are mainly affected by type of interfering peptides as well as concentration of each peptide and GLP in the environmental samples.