A microfluidic paper-based analytical device (µPAD) has been developed for rapid detection of Staphylococcus aureus in resource limited settings. Staphylococcus aureus can be detected from food or clinical samples with enrichment in Sterile PYP broth for 1.5 h and used for detection of Alkaline Phosphatase activity using chromogenic substrate Blue Phos. The test is accompanied by Gram staining of the sample and coagulase test. Qualitative detection is based on visual detection while quantitative analysis is done using ImageJ TM software. The test can detect up to 10 4 CFUmL-1 of Staphylococcus aureus within 3-4 h. This test is cost effective, does not need any trained personnel or sophisticated instruments for analysis. Analysis can be done on a smart phone. The test is ideal for presumptive diagnosis of Staphylococcus aureus for clinicians operating in remote locations and can be used as a screening test by food industries.