The performance of matrix assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF) of bacterial proteins strongly depends on sample preparation. It is found that the mass spectral profiles obtained from direct MALDI-TOF MS of the protein extracts are much weaker for individual bacterial cells than compared to those prepared by the Tris-EDTA buffer approach (TEBA). Characteristic mass spectral peaks were observed in the mass range from 3,000 to 15,000 Da. The mass peaks reported earlier and claimed to serve as species-specific biomarkers are consistently found here as well. Mass peaks at m/z of 3636, 5466, 5750, 6315, 6547, 7274, 9192, and 9742 are found for Escherichia coli studied and assigned as specific biomarkers. Similarly, specific mass peaks have been identified at m/z 5443, 7270, 7724, and 9888 for Bacillus subtilis, and at 3603, 5496, 6800, 8858 and 9531 for Serratia marcescens. The detection limits for the three target bacteria range from 2.4 × 105 to 3.3 × 105 cfu·mL-1. We conclude that the TE buffer approach can produce reliable data for rapid classification, high-resolution and highly sensitive detection of bacteria.