BACKGROUND With infiltration, high-grade glioma easily causes the boundary between tumor tissue and adjacent tissue to become unclear and results in tumor recurrence at or near the resection margin according to the incomplete surgical resection. Fourier transform infrared spectroscopy (FTIR) technique has been demonstrated to be a useful tool that yields a molecular fingerprint and provides rapid, nondestructive, high-throughput and clinically relevant diagnostic information. MATERIAL AND METHODS FTIR was used to investigate the morphological and biochemical properties of human astrocytes (HA), microglia (HM1900), glioma cells (U87), and glioblastoma cells (BT325) cultured in vitro to simulate the infiltration area, with the use of multi-peak fitting and principal component analysis (PCA) of amide I of FTIR spectra and the use of hierarchical cluster analysis (HCA). RESULTS We found that the secondary structures of the 4 types of cells were significantly different. The contents of a-helix structure in glial cells was significantly higher than in the glioma cells, but the levels of ß-sheet, ß-turn, and random coil structures were lower. The 4 types of cells could be clearly separated with 85% for PC1 and 12.2% for PC2. CONCLUSIONS FTIR can be used to distinguish between human astrocytes, microglia, glioma, and glioblastoma cells in vitro. The protein secondary structure can be used as an indicator to distinguish tumor cells from glial cells. Further tissue-based and in vivo studies are needed to determine whether FTIR can identify cerebral glioma.