Display omitted •Integrated standardization of propolis by chromatographic, spectroscopic, biological and palynological analyses was executed.•A dual visualization approach with four image analysis software adopting different algorithms were comparatively evaluated.•Multivariate analysis was used to discriminate between propolis types and to find health-relevant phytoconstituents.•HPTLC-ESI-MS were utilized for precise identification of the discriminatory phytoconstituents.•Pollen analysis was implemented to determine the botanical origin of Egyptian propolis for the first time. An integrated method for standardization of propolis using chromatographic, spectroscopic, palynological and biological analyses was implemented in this study. Digitally-enhanced HPTLC images using dual visualization approach with four image analysis software packages adopting different digitalization algorithms were comparatively evaluated i.e.; Sorbfil TLC View®, ImageJ®, JustTLC® and Gel Analyzer®. ImageJ® and Gel Analyzer® showed superior figures of merits in case of flavonoids and phenolic acids quantitation, respectively. Unsupervised and supervised multivariable pattern recognition models were constructed for comprehensive discrimination between the three propolis types (blue, orange and green) collected from various geographical locations worldwide. HPTLC-ESI-MS was utilized for precise identification of the discriminatory phytoconstituents. Fingerprint-efficacy relationship analysis was conducted via an Orthogonal Projection to Latent Structure multivariate model to unravel the bio-efficient markers in terms of α-glucosidase and α-amylase inhibition as main targets. 3,4-Dimethoxycinnamic acid, caffeic acid, isoferulic acid, rosmarinic acid, and quercetin were found to be the main health-relevant markers. A complementary fast, simple and readily available UV spectroscopic method using aluminium chloride as bathochromic shift reagent was used as an independent method for prediction of aforementioned biomarkers using a validated Partial Least Squares Regression model. In addition, palynological analysis was implemented to determine the botanical origin of Egyptian propolis for the first time. Twenty-eight pollen types assigned to 13 families were identified, with Asteraceae being the highest representative one. The investigated samples lacked dominant pollen types, which reflected the multifloral origin of Egyptian propolis. Identification of plant sources of propolis, which directly affect its chemical composition and subsequently its biological efficacy represents an integral part of its standardization.