Here, we report on the development of a smartphone-based application (app) for the accurate and sensitive measurement of hemoglobin (Hb). Hb values obtained using the app correlated significantly (R² = 0.976) with those obtained by an automated hematology analyzer. Bland–Altman analysis showed that the standard deviation of the difference between Hb measured by the two methods was 0.35 g dL⁻¹ and the limits of agreement between the two methods were −0.62 to 0.75. A limit of detection of 0.66 g dL⁻¹ was achieved using the app along with a 2 g dL⁻¹ limit of quantification. Furthermore, measurement using the app was highly sensitive (94%) and specific (90%). The app was developed for a smartphone and it will be particularly useful in resource-limited conditions for the rapid, accurate, highly sensitive and specific assessment of anemia.