Traditional methods for the microorganisms identification are based on the use of strategies such culture media, isolation in selective media, biochemical identification and serological characterization.These strategies are expensive, have low specificity, low sensitivity and are time consuming.In recent years, different molecular strategies such as the Polymerase Chain Reaction (PCR) have been developed, especially quantitative real-time PCR (qPCR) for Salmonella spp.and Escherichia coli detection and quantification in different matrices.These molecular methods are simple, reproducible, flexible and allow a rapid identification.This chapter presents an analysis about methods for Salmonella spp.and E. coli detection, and make emphasis on the use of qPCR and its standardization aimed at the diagnosis of these public health importance microorganisms.