Bees mainly rely on pollen for their protein resources. As these molecules are essential for numerous aspects of bee physiology like ovary development and larval growth, their quantification and determination are crucial to evaluate diet quality. However, the term “protein” has been used to mention crude protein, total amino acids, or protein sensu stricto (i.e., polypeptides of molecular weight >10,000 Da). In addition to this ambiguity, current methods for protein quantification suffer from bias due to nonprotein nitrogen and protein-to-protein variations. A reliable and nondestructive method to quantify the pollen polypeptides is then essential to estimate bee food source. The present paper aims (a) to detail such a protocol, (b) to evaluate its efficiency, and (c) to confront its results to those returned by traditional methods of protein estimation. Our protocol clearly overrides some bias of previous methods and is highly reliable. Results show the high variability in content of pollen polypeptides and suggest that the main part of the proteinaceous nitrogen is from oligopeptides. They also highlight that hand-collected pollen is a better matrice than pollen loads to estimate the polypeptides of pollen as bee food source.