For the purpose of this study, we used an extraction protocol recommended for clinical laboratories for MALDI-TOF MS profiling of bacteria. Seventeen closely related strains of the Lactobacillus acidophilus group were cultivated under various growth conditions (growth medium, time, and temperature) and analyzed. Out of a total of 327 samples, 80 % were correctly assigned to the species level and 13 % only to the genus level. When using data obtained from strains cultured for lengthy periods (7 days), the identification success rate was reduced due to poor signal quality, whereas with shorter cultivation times there was no influence of growth conditions on the assignment of particular strains to their corresponding species. However, variations in certain cultivation parameters were found to influence identification and differentiation of most of the examined strains. Strain discrimination was frequently found to be dependent on the selection of culture conditions. MALDI-TOF MS data treatment (strain-specific peak detection, BioTyper scoring, subtyping, or cluster analysis) also contributed to the discriminatory power of the method.