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Mitochondrial transfer to recipient cells triggers a respiratory burst by increasing ATP production and cellular energy metabolism. However, its impact on intracellular metabolic shifts remains unclear. This study introduces a novel methodological approach and new biological insights into mitochondrial dynamics in cancer cells. We developed fluorescence-lifetime imaging microscopy (FLIM) intensity-based image segmentation (FIBIS), an algorithm optimized for single-mitochondrion analysis. FIBIS utilizes NADH autofluorescence, eliminating the need for biomarker staining, and improves mitochondrial detection accuracy by 35% compared to raw intensity thresholding. This method is particularly effective for analyzing dynamic mitochondria in live cells. Using FIBIS, we show that normal epithelial mitochondria uptake alters the free NADH-to-bound NADH ratio, increasing bound NADH in both estrogen- and progesterone receptor-positive and triple-negative breast cancer cells. Additionally, mitochondrial transfer enhances cancer cell sensitivity to oxidative stress-inducing anti-cancer drugs, suggesting a potential restoration of normal reactive oxygen species tolerance. Overall, FIBIS is a robust methodological approach that uses the phasor-FLIM technique to analyze NADH levels (free and bound) at the single-mitochondrion level, providing new biological insights into transferred mitochondrial dynamics in cancer cells.