In acute myeloid leukemia, the burden of CD34+CD38- leukemia stem cells (LSC) has prognostic value at diagnosis and after induction chemotherapy. Since different methods of LSC quantification have been proposed, we determined the prognostic value on overall survival and incidence of relapse of these methods across ELN2017 risk groups, using data from the HOVON-SAKK132 trial. In addition, we have evaluated the optimal number of acquired white blood cells for accurate LSC detection and the prognostic value of individual LSC markers. Results show that acquiring 1 million white blood cells is essential for accurate LSC-negativity assessment. Among different LSC markers, CD44 overexpression on CD34+CD38- cells was the only insignificant marker in our panel. Testing the impact of several published variations on the analysis for LSC assessments on prognostic value for overall survival and cumulative incidence of relapse, showed marginal differences, demonstrating the robust prognostic value of LSC burden. For further clinical implementation, the optimal LSC assessment may differ among ELN risk groups. In conclusion, LSC burden is a robust prognostic factor and insight in the different methods of LSC definition can facilitate the clinical implementation.