The measurement of hemoglobin concentration ([Hb]) is performed routinely as a part of a complete blood cell count to evaluate the oxygen-carrying capacity of blood. Devices currently available to physicians and clinical laboratories for measuring [Hb] are accurate, operate on small samples, and provide results rapidly, but may be prohibitively expensive for resource-limited settings. The unavailability of accurate but inexpensive diagnostic tools often precludes proper diagnosis of anemia in low-income developing countries. Therefore, we developed a simple paper-based assay for measuring [Hb]. A 20-μL droplet of a mixture of blood and Drabkin reagent was deposited onto patterned chromatography paper. The resulting blood stain was digitized with a portable scanner and analyzed. The mean color intensity of the blood stain was used to quantify [Hb]. We compared the performance of the paper-based Hb assay with a hematology analyzer (comparison method) using blood samples from 54 subjects. The values of [Hb] measured by the paper-based assay and the comparison method were highly correlated (R(2) = 0.9598); the standard deviation of the difference between the two measurements was 0.62 g/dL. The assay was accurate within 1 g/dL 90.7% of the time, overestimating [Hb] by ≥1 g/dL in 1.9% and underestimating [Hb] by ≥1 g/dL in 7.4% of the subjects. This study demonstrates the feasibility of the paper-based Hb assay. This simple, low-cost test should be useful for diagnosing anemia in resource-limited settings, particularly in the context of care for malaria, HIV, and sickle cell disease patients in sub-Saharan Africa.