Mass spectrometry was used to monitor neutral chemical species from sugar cane bagasse that could volatilize during the bagasse ozonation process. Lignin fragments and some radicals liberated by direct ozone reaction with the biomass structure were detected. Ozone density was monitored during the ozonation by optical absorption spectroscopy. The optical results indicated that the ozone interaction with the bagasse material was better for bagasse particle sizes less than or equal to 0.5 mm. Both techniques have shown that the best condition for the ozone diffusion in the bagasse was at 50% of its moisture content. In addition, Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM) were employed to analyze the lignin bond disruptions and morphology changes of the bagasse surface that occurred due to the ozonolysis reactions as well. Appropriate chemical characterization of the lignin content in bagasse before and after its ozonation was also carried out.