Graphite oxide synthesis dynamics were investigated using a sampling technique. The synthesis of graphite oxide was carried out by a modified Hummers’ method. Small samples of the solid phase (30–50 mg) were collected from the reaction mixture and analyzed by thermogravimetric analysis, differential scanning calorimetry, scanning electron microscopy, X-ray diffraction, Raman spectroscopy, energy-dispersive X-ray spectroscopy, and X-ray photoelectron spectroscopy. The strongest oxidation was detected 10 min after the start of the synthesis, i.e., after the addition of KMnO4, when the formation of the graphite oxide phase with intercalated guest molecules begins. The intercalation of graphite started after 30 min of synthesis when the temperature was increased to 35 °C. The addition of ice into the reaction mixture leads to the increase in the COOH group concentration, whereas the concentration of C[dbnd]O groups slightly changes, and the concentration of the C–O and C[dbnd]O groups remains almost constant. It was found that the degree of oxidation of graphite oxide exhibited complex change, and H2O2 plays a significant role not only in the removal of impurities but also in the increase in the GO oxidation degree that is reflected by a higher concentration of oxygen-containing functional groups. Differential scanning calorimetry and thermogravimetric analysis data confirmed that the additions of ice and H2O2 induce the stronger formation of surface functional groups instead of intercalated guest species.