TMAH-based Py-MS has been investigated to apply for a real-time classification of biological agents in the field. Acquiring reproducible data from mass spectrometry is a key to biological detection in the field. Nevertheless, it has been little studied on what factors could affect to the reproducibility of the TMAH-based Py-MS spectrum patterns. Given the TMAH-based Py-MS applied to the field system, several factors which could affect to the reproducible pattern of TMAH-based Py-MS spectra are needed to be examined, including changes in TMAH injection volume, growth temperature for microorganism, and number of cells collected in pyrolyzer, and implication of stabilizer used for lyophilization. This study showed that the reproducibility of the spectrum patterns was significantly hindered by changes in TMAH concentration and cell number, and stabilizer implication but not by growth temperature. Among those at low TMAH concentration(0.015m) was not observed the significant alterations of the spectrum pattern even when its injection volume was changed, yet was in different cell numbers and stabilizer implication.