The identification of Salmonella which is a major food-borne pathogen generally takes more than 2-3 days. More reliable, rapid and cost effective results can be achieved with MALDI-TOF which is based on mass spectrometry principles in comparison with the automated systems in routine microbiology laboratories. The aim of this study is to evaluate the use of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF) in gastrointestinal infections caused by those pathogen microorganisms by using practical cost-effective and reliable methods within a less time period. Between August 2007 and August 2011, 11186 stool specimens of patients were included to the study. Patient samples were routinely inoculated onto CHROMagar Salmonella plus and Mac Conkey agar media. 347 Salmonella species were run with both Phoenix automated system and MALDI-TOF. The results of both of the methods are 100% concordant with each other. Using Chromogar Salmonella plus media together with MALDI-TOF identification shortened the time, helped to detect Salmonella species easily. Species identifications are being performed in both of the systems, but identification of subspecies still require conventionally serological typing with Salmonella antiserums. MALDI-TOF will help the routine clinical microbiology laboratory to detect Salmonella rapidly with only one media and in 48 h together with their antibiotic susceptibility testings in a cost-effective and reliable manner.