Non-destructive qualitative and quantitative characterization of a radiosensitizer, 5-iodo-2'-deoxyuridine (IdUrd), incorporated within injectable microspheres of a biodegradable polymer, poly(D,L-lactide-co-glycolide) (PLGA), was performed using Fourier transform (FT) Raman spectroscopy. Raman spectra of IdUrd, free and entrapped in microspheres, were recorded under fluorescence-free conditions, described and assigned. For the Raman bands of the PLGA microspheres, assignments with preferential localization of the corresponding vibrations at lactic or glycolic units were proposed. No evidence for drug-polymer interactions in microspheres was found. This allowed the FT-Raman spectra to be used for the quantification of the IdUrd content in the samples. For the microspheres with IdUrd loadings varying from 2 to 27% of the total weight, the methodology used provided good reproducibility and precision (1%). Within the sensitivity of the technique, samples exposed to sterilization doses (27 kGy) of gamma-radiation did not exhibit marked changes in the drug structure.