We designed an in situ mass spectrometry (in situ MS) analysis method and developed to identify the products of glucose decomposition under hydrothermal condition for the first time. The in situ MS analysis was performed by coupling a tubular batch reactor with a quadrupole mass analyzer via custom-built connection fittings. The products of glucose decomposition were investigated by in situ MS, mass spectrometry of cold effluent, and high-performance liquid chromatography (HPLC) analysis of cold effluent and the results were compared. At 140 °C, in situ MS and mass spectrometry of cold effluent showed that the decomposition of glucose does not proceed; this was confirmed by comparison with the mass spectral database for glucose. At 180 °C or higher, a clear base fragmentation peak of 5-hydroxymethylfurfural (5-HMF) at position m/z 97 and that of furfural at m/z 96, formic acid (m/z=46) and levulinic acid (m/z=116) were observed by mass spectrometry. No levulinic acid or furfural was observed through conventional HPLC analysis under any condition; only glucose, formic acid, and 5-HMF could be detected. The effectiveness of in situ MS analysis is clear, compared to mass spectrometry analysis of cold effluent and HPLC analysis.