This study was to demonstrate the application of spectrophotometry for rapid and presumptive differentiation of Salmonella from other Enterobacteriaceae based on lysine decarboxylase (LDC) activity. The pH indicator system and cocktails of Salmonella selective inhibitors were optimized to derive a new selective broth (patent pending no. 1301006577) for Salmonella screening. Phenol red (PR) was the most suitable pH indicator for the lysine decarboxylase broth and then derived a new medium, developed lysine decarboxylase broth with phenol red (dLDBPR). This enrichment medium was able to produce the highest absorbance to differentiate LDC-positive from -negative samples at 587 nm wavelength and proven to be rapid and useful approach to identify salmonellae and Enterobacteriaceae from other competitors. The variation of bacterial growth and broth pH generated from the glucose fermentation and LDC activity resulted in broth color changes. The most efficient broth was dLDBPR with RVS inhibitors (dLRVS) enabling fast positive-color shift within 8-10 h and sustained cell viability in all LDCpositive Salmonella samples. However, dLRVS treatment contained some false LDC-positive results in some samples with non-Salmonella competitors (Escherichia coli and Klebsiella pneumoniae). Further optimization of selective inhibitors towards Salmonella detection is in progress. The assay was proven to be rapid and useful approach to distinguish salmonellae and Enterobacteriaceae from other competitors.