Propolis is a resinous substance collected by honeybees from leaf buds and cracks in the bark of various plants, and it is composed of 50% resin (composed of flavonoids and related phenolic acids), 30% wax, 10% essential oils, 5% pollen and 5% various organic compounds. Propolis cannot be used as raw material, and it must be purified by extraction with solvents. This process should remove the inert material and preserve the polyphenolic fraction, which is considered to contribute more to the observed healing effects than the other propolis constituents. Therefore, the assay of propolis polyphenols is of interest, and this paper describes the results obtained in the analysis of propolis by means of a gradient HPLC or mass spectrometry. HPLC in the gradient mode and coupled with photodiode array detection remains the method of choice for the assay of most relevant components of propolis. Direct analysis by APCI-IT-MS represents a valuable alternative to obtain typical fingerprints of propolis and a reliable identification of a large number of propolis components.