Aim:Identification of pathogenic clinical bacterial isolates is mainly dependent on phenotypic and genotypic characteristics of the microorganisms. These conventional methods are costive, time-consuming, and need special skills and training. An alternative, mass spectral (proteomics) analysis method for identification of clinical bacterial isolates has been recognized as a rapid, reliable, and economical method for identification. This study was aimed to evaluate and compare the performance, sensitivity and reliability of traditional bacteriology, phenotypic methods and matrix-assisted laser desorption-ionization-time-of-flight mass spectrometry (MALDI-TOF MS) in the identification of clinical Escherichia coli and Salmonella isolates recovered from chickens.Materials and Methods:A total of 110 samples (cloacal, liver, spleen, and/or gall bladder) were collected from apparently healthy and diseased chickens showing clinical signs as white chalky diarrhea, pasty vent, and decrease egg production as well as freshly dead chickens which showing postmortem lesions as enlarged liver with congestion and enlarged gall bladder from different poultry farms.Results:Depending on colonial characteristics and morphological characteristics, E. coli and Salmonella isolates were recovered and detected in only 42 and 35 samples, respectively. Biochemical identification using API 20E identification system revealed that the suspected E. coli isolates were 33 out of 42 of colonial and morphological identified E. coli isolates where Salmonella isolates were represented by 26 out of 35 of colonial and morphological identified Salmonella isolates. Serological identification of isolates revealed that the most predominant E. coli serotypes were O1 and O78 while the most predominant Salmonella serotype of Salmonella was Salmonella Pullorum. All E. coli and Salmonella isolates were examined using MALDI-TOF MS. In agreement with traditional identification, MADI-TOF MS identified all clinical bacterial samples with valid scores as E. coli and Salmonella isolates except two E. coli isolates recovered from apparently healthy and diseased birds, respectively, with recovery rate of 93.9% and 2 Salmonella isolates recovered from apparently healthy and dead birds, respectively, with recovery rate of 92.3%.Conclusion:Our study demonstrated that Bruker MALDI-TOF MS Biotyper is a reliable rapid and economic tool for the identification of Gram-negative bacteria especially E. coli and Salmonella which could be used as an alternative diagnostic tool for routine identification and differentiation of clinical isolates in the bacteriological laboratory. MALDI-TOF MS need more validation and verification and more study on the performance of direct colony and extraction methods to detect the most sensitive one and also need using more samples to detect sensitivity, reliability, and performance of this type of bacterial identification.