Background <p>High sensitivity to mitochondria damage belongs to established differences between stem cells of acute myeloid leukemia (AML) and their healthy counterparts. Mitophagy, as the key mitochondria quality control, is a possible therapy target in AML. However, research in this field requires simple and robust methods for functional measurement of mitochondria clearance.</p> Methods <p>We developed protocols to analyze (i) the rate of mitochondria clearance and (ii) total mitochondrial mass and mitochondria polarization using MitoTracker dyes and flow cytometry. Substantial improvement was achieved by incorporating (i) a proliferation dye to account for the effect of cell division on mitochondria content and (ii) reference cells to compensate for variable cell density. These protocols were tested in 12 leukemia cell lines with different variants of the tumor suppresor <i>TP53,</i> as well as the adherent cell line HS-5.</p> Results <p>Repeated analyses confirmed high sensitivity and reproducibility of our protocols. The mitophagy rate in p53-null cells was higher compared to cells with the wild-type p53. Mitophagy inhibition was associated with an increase of the total mitochondria mass. The impact of mitophagy inhibition on mitochondria polarization was context-dependent: mitochondria depolarization was associated with cell death induction whereas hyperpolarization was observed during cell recovery in resistant cell lines.</p> Conclusion <p>Our optimized protocols represent a useful tool for analysis of mitophagy and mitochondria content in various cell types.</p>