Foodborne pathogens are a major public health threat and financial burden for the food industry, individuals, and society, with an estimated 76 million cases of food-related illness occurring in the United States alone each year. Three of the most important causative bacterial agents of foodborne diseases are pathogenic strains of Escherichia coli , Salmonella spp., and Listeria monocytogenes , due to the severity and frequency of illness and disproportionally high number of fatalities. Their continued persistence in food has dictated the ongoing need for faster, simpler, and less expensive analytical systems capable of live pathogen detection in complex samples. Culture techniques for detection and identification of foodborne pathogens require 5-7 days to complete. Major improvements to molecular detection techniques have been introduced recently, including polymerase chain reaction (PCR). These methods can be tedious; require complex, expensive instrumentation; necessitate highly trained personnel; and are not easily amenable to routine screening. Here, a paper-based analytical device (μPAD) has been developed for the detection of E. coli O157:H7, Salmonella Typhimurium, and L. monocytogenes in food samples as a screening system. In this work, a paper-based microspot assay was created by use of wax printing on filter paper. Detection is achieved by measuring the color change when an enzyme associated with the pathogen of interest reacts with a chromogenic substrate. When combined with enrichment procedures, the method allows for an enrichment time of 12 h or less and is capable of detecting bacteria in concentrations in inoculated ready-to-eat (RTE) meat as low as 10(1) colony-forming units/cm(2).