Polyethylene glycol (PEG) has widespread applications in pharmacy, medicine, and cosmetics, but its biological degradation is not completely understood, especially because of the complex analytics required for determining changes in molar mass and side or end groups, respectively. Here, we describe the activity of Pseudomonas sp. on PEG with molar masses of 6,000, 20,000 and 40,000 g mol-1. The molar mass distribution after degradation was analyzed by size-exclusion chromatography (SEC) with different detection methods as well as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and the differences between the results demonstrated. Further, the influence of the complex bacterial cultivation medium on analysis results was discussed. MALDI-TOF MS analysis revealed hydroxyethylene end-units on the PEG-terminus after bacterial degradation providing hints on the bacterial degradation pathway via end-unit scissioning followed by oxidation of the cleaved unit.