BACKGROUND The quantification of fetal cells in the maternal circulation remains an important goal to determine the amount of anti-D necessary to prevent active immunization of a D- mother giving birth to a D+ baby. Underestimation of fetomaternal hemorrhage (FMH) results in inefficient anti-D prophylaxis and maternal immunization; overestimation of FMH results in higher doses of passively transferred anti-D, higher costs, and the risk of disease transmission. Thus, a reliable method to quantitatively assess FMH is necessary. STUDY DESIGN AND METHODS Serial dilutions of artificial FMH were quantitatively measured by three different methods: flow cytometry, fluorescence microscopy (each after anti-D staining), and by the Kleihauer-Betke test. The accuracy and precision of the three methods were compared by statistical analysis. RESULTS Fluorescence microscopy and flow cytometry were comparably accurate and precise in quantifying FMH. In contrast, the accuracy of the Kleihauer-Betke test was poor, resulting in substantial overestimation of FMH in the samples with lower fetal cell concentrations. CONCLUSION Anti-D flow cytometry and fluorescence microscopy for detection of fetal cells offer equally reliable and precise methods in contrast to the Kleihauer-Betke test. Fluorescence microscopy may be established as standard to quantify FMH in clinical practice because it is comparable to flow cytometry; in addition, it is time saving and is less expensive.