This paper reports the characterization of the biodegradation intermediates of octylphenol octaethoxylate (OP8EO) by means of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The biodegradation test study was carried out in a pure culture (Pseudomonas putida S-5) under aerobic conditions using OP8EO as the sole carbon source and O-18-labeled water as an incubation medium. In the MALDI-MS spectra of biodegraded samples, a series of OPnEO molecules with n = 2-8 EO units and their corresponding carboxylic acid products (OPnEC) were observed. The use of purified OP8EO enabled one to distinguish the shortened OPEO molecules as biodegradation intermediates. Furthermore, the formation of OP8EC (the oxidized product of OP8EO) supported the notion that terminal oxidation is a step in the biodegradation process. When biodegradation study was carried out in O-18-labeled water, incorporation of O-18 atoms into the carboxyl group was observed for OPEC, while no incorporation was observed for the shortened OPEO products. These results could provide some rationale to the biodegradation mechanism of alkylphenol polyethoxylates.