The purpose of this study was to evaluate the quantification of fetomaternal hemorrhage by the manual and automated microscopic analysis of Kleihauer-Betke stained slides and by flow cytometry. Blood smears were stained and evaluated manually according to the Kleihauer-Betke test. The same slides were used for automated microscopy. In addition, blood flow cytometry was performed by anti-hemaglobin F immunostaining. Fetomaternal hemorrhage >0.1% was detected in 4 patients by manual and automated Kleihauer-Betke test and by blood flow cytometry. Fetomaternal hemorrhage was absent according to all 3 methods in 13 patients; fetomaternal hemorrhage<0.1% was detected in 27 patients by either manual or automated Kleihauer-Betke test or both. Moderate agreement was observed between the manual and automated Kleihauer-Betke test (weighted kappa, 0.56; 95% CI, 0.33-0.78). Agreement between the manual Kleihauer-Betke test and blood flow cytometry was fair (weighted kappa, 0.40; 95% CI, 0.15-0.66). Automated microscopic detection of fetal blood cells in clinical samples provides accurate quantification that is comparable to the manual Kleihauer-Betke test in both small and large fetomaternal hemorrhage. Blood flow cytometry is capable only of quantifying fetomaternal hemorrhage of >0.1%.