Diagnosis of dengue infection during the febrile stage has been challenging. We evaluated the accuracy of NS1 protein detection in diagnosing dengue infection in patients presenting with acute febrile illness in Bangkok, Thailand. Of the 235 subjects presented with fever of unknown source within 5 days, 132 (56.2%) were male with the median age of 17.8 (range, 3-52) years. The median duration of fever was 4 (range, 1-5) days. One hundred seventy-one (72.8%) patients had dengue infection, of which 158 (92.4%) were secondary infections. The sensitivity of NS1 Ag test was 63.2% (95% confidence interval [CI], 55.7-70.0), and the specificity was 98.4% (95% CI, 91.7-99.7). The positive and negative predictive values were 99.0% and 52.5%, respectively. The immune complex dissociation by acid treatment increase sensitivity from 63.2% to 72%. In an endemic area, Plateliatrade mark NS1 Ag test has limited sensitivity but very high specificity for diagnosis of dengue infection in patients with acute febrile illness.