Cell counter-based formulas have been used in the differential diagnosis of microcytic anemia. The measurement of RBC subpopulations is now available on the Sysmex XE 5000 analyzer (Sysmex, Kobe, Japan). We describe the new formulas: % microcytic - % hypochromic; and % microcytic - % hypochromic - red cell distribution width (RDW), derived from the percentages of microcytic and hypochromic RBCs. The present study aimed to prospectively evaluate the reliability of these new formulas in the differential diagnosis of microcytosis and β-thalassemia screening compared with already published indices. The indices were calculated for a set of 250 iron-deficient patients and 270 β-thalassemia carriers. Independent samples t test and receiver-operating characteristics analysis were applied. The % microcytic - % hypochromic - RDW, % microcytic - % hypochromic, and Green and King indices provided higher areas under the curve. The % microcytic - % hypochromic - RDW was the most reliable index evaluated, with 100% sensitivity and 92.6% specificity. This index can be used to efficiently screen patients with microcytosis for further hematologic studies to confirm β-thalassemia.