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The extraction and analysis of polyphenols from raw propolis was performed.A closed-vessel microwave-assisted extraction (MAE) was optimized for the first time.The HPLC analysis of propolis extracts was carried out on a fused-core C18 column.The developed method was fully validated in agreement with international guidelines.Great variability was observed in the polyphenol content of propolis extracts.A closed-vessel microwave-assisted extraction (MAE) technique was optimized for the first time for the extraction of polyphenols from raw propolis. The results obtained by means of response surface experimental design methodology showed that the best global response was reached when the extraction temperature was set at 106 °C, the solvent composition close to EtOH–H2O 80:20 (v/v), with an extraction time of 15 min. In comparison with other techniques, such as maceration, heat reflux extraction (HRE) and ultrasound-assisted extraction (UAE), the extraction with MAE was improved by shorter extraction time and lower volume of solvent needed.The HPLC analyses of propolis extracts were carried out on a fused-core Ascentis Express C18 column (150 mm × 3.0 mm I.D., 2.7 μm), with a gradient mobile phase composed by 0.1% formic acid in water and acetonitrile. Detection was performed by DAD and MS.The method validation indicated that the correlation coefficients were >0.999; the limit of detection was in the range 0.5–0.8 μg/ml for phenolic acids and 1.2–3.0 μg/ml for flavonoids; the recovery range was 95.3–98.1% for phenolic acids and 94.1–101.3% for flavonoids; the intra- and inter-day %RSD values for retention times and peak areas were ≤0.3 and 2.2%, respectively.The quali- and quantitative analysis of polyphenols in Italian samples of raw propolis was performed with the validated method. Total phenolic acids ranged from 5.0 to 120.8 mg/g and total flavonoids from 2.5 to 168.0 mg/g.The proposed MAE procedure and HPLC method can be considered reliable and useful tools for the comprehensive multi-component analysis of polyphenols in propolis extracts to be used in apitherapy.