Anemia affects a quarter of the world's population, and a lack of appropriate diagnostic tools often prevents treatment in low-resource settings. Though the HemoCue 201+ is an appropriate device for diagnosing anemia in low-resource settings, the high cost of disposables ($0.99 per test in Malawi) limits its availability. We investigated using spectrophotometric measurement of blood spotted on chromatography paper as a low-cost (<$0.01 per test) alternative to HemoCue cuvettes. For this evaluation, donor blood was diluted with plasma to simulate anemia, a micropipette spotted blood on paper, and a bench-top spectrophotometer validated the approach before the development of a low-cost reader. We optimized impregnating paper with chemicals to lyse red blood cells, paper type, drying time, wavelengths measured, and sensitivity to variations in volume of blood, and we validated our approach using patient samples. Lysing the blood cells with sodium deoxycholate dried in Whatman Chr4 chromatography paper gave repeatable results, and the absorbance difference between 528 nm and 656 nm was stable over time in measurements taken up to 10 min after sample preparation. The method was insensitive to the amount of blood spotted on the paper over the range of 5 μL to 25 μL. We created a low-cost, handheld reader to measure the transmission of paper cuvettes at these optimal wavelengths. Training and validating our method with patient samples on both the spectrometer and the handheld reader showed that both devices are accurate to within 2 g dL(-1) of the HemoCue device for 98% and 95% of samples, respectively.