Background <p>Cardiolipin (CL) is a signature phospholipid of mitochondria that maintains the integrity of mitochondrial membrane and supports proper mitochondrial function. Alterations in CL level and composition can impair or, conversely, improve mitochondrial function and bioenergetics, both of which are critical for cancer metabolism. However, conflicting reports on CL levels across different cancer types and limited research using human patient samples limit our understanding of its diagnostic potential.</p> Methods <p>This cross-sectional study explores CL concentrations in gastric and colon cancer tissues using a CL-specific fluorescent probe MitoCLue and compares them to adjacent healthy tissues.</p> Results <p>In gastric cancer, CL levels showed no significant differences between tumor and healthy tissues, suggesting that metabolic shifts in gastric cancer do not affect total CL content. In contrast, colon cancer tissues exhibited a significant 33% increase in CL levels, indicating mitochondrial adaptation and/or increase in mitochondrial mass in colon cancer. No associations were found between CL levels and patient demographic factors; although a weak correlation with body mass index was noted.</p> Conclusion <p>We successfully applied MitoCLue to quantitatively assess the total CL level in healthy and tumor tissues from patients with gastric or colon cancer. The distinct CL levels in gastric and colon cancer suggest that there are cancer-type specific mitochondrial adaptations, reflecting unique bioenergetic demands and metabolic reprogramming pathways. While a 33% increase in CL levels was observed in colon cancer tissues compared to healthy adjacent tissues, this modest variation may limit its utility as a standalone biomarker.</p>