Strontium Titanate (STO) substrates were studied by electron paramagnetic resonance (EPR) spectroscopy to assess possible changes incurred by deposition of multiferroic thin films. To this effect, STO was vacuum annealed at pressures of 10−5 Torr for one hour at temperatures in the range of 200 – 500 °C. EPR spectra, measured before and after each anneal, revealed changes in the amount of three different defects, Cr3+, Fe3+ and an iron-oxygen vacancy complex, Fe3+Vo. The latter was used to monitor the diffusion of oxygen. EPR analysis showed that Fe3+Vo increases from its as-grown value, suggesting that a charged oxygen species is mobile in the substrate under film deposition conditions. Coupled with a subsequent O2 anneal showing minimal change in the Fe3+Vo signal, the data indicate a loss of oxygen from the sample during vacuum annealing. As charged oxygen vacancies may affect the substrate as well as the substrate/ thin film interface, these results are important for understanding the behavior of multiferroic devices built on STO substrates.