Polybutadienes (PB) and polyisoprenes (PI) with various molecular weights and polydispersities were epoxidized applying two different synthetic routes. The degree of epoxidation was determined by means of matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). It has been found that the degree of functionalization of polybutadiene is generally lower than that for polyisoprenes. Additionally, PB shows lower degrees of epoxidation at higher masses, whereas the level of functionalization of PI was unaffected by the molar mass. Coupling of gel permeation chromatography (GPC) with MALDI-TOF mass spectrometry was successfully used for the investigation of epoxidized polymers with higher masses. Using mass spectra of single fractions, the degree of epoxidation could be determined and applied for a calibration of GPC.