New Zealand mānuka (Leptospermum scoparium) honey is a premium food product. Unfortunately, its high demand has led to “not true to label” marketed mānuka honey. Robust methods are therefore required to determine authenticity. We previously identified three unique nectar-derived proteins in mānuka honey, detected as twelve tryptic peptide markers, and hypothesized these could be used to determine authenticity. We invoked a targeted proteomic approach based on parallel reaction-monitoring (PRM) to selectively monitor relative abundance of these peptides in sixteen mānuka and twenty six non-mānuka honey samples of various floral origin. We included six tryptic peptide markers derived from three bee-derived major royal jelly proteins as potential internal standards. The twelve mānuka-specific tryptic peptide markers were present in all mānuka honeys with minor regional variation. By comparison, they had negligible presence in non-mānuka honeys. Bee-derived peptides were detected in all honeys with similar relative abundance but with sufficient variation precluding their utility as internal standards. Mānuka honeys displayed an inverse relationship between total protein content and the ratio between nectar- to bee-derived peptide abundance. This trend reveals an association between protein content on possible nectar processing time by bees. Overall, these findings demonstrate the first successful application of peptide profiling as an alternative and potentially more robust approach for mānuka honey authentication.