Background Carica papaya L. is a dioecious plant with leaves known for their medicinal and nutritional value due to their rich phytochemical content, including flavonoids. Phytochemical variability arising from plant maturity and sex differences may influence the bioactivity of the plant. Objectives This research aims to investigate the variations of flavonoids in C. papaya leaves across different maturation stages and the impact of sex differences on the accumulation of these compounds. Methods An ultra-high performance liquid chromatography–tandem mass spectrometry method was developed to quantify 6 flavonoids (manghaslin, clitorin, rutin, nicotiflorin, quercetin, and isorhamnetin) in the leaves simultaneously. The acquired data were further analyzed using chemometric tools. Results Significantly higher concentrations of flavonoids were observed in shoots and young leaves in comparison to older leaves. Furthermore, distinct variations in flavonoid composition between male and female plants were also observed, especially in young leaves, as evidenced by hierarchical clustering analysis. Further analysis of flavonoid content indicates that clitorin, nicotiflorin, and rutin could serve as chemical markers to differentiate maturation stages and sexes. Conclusion The study revealed significant variations in flavonoid concentrations during leaf development and between male and female plants, highlighting the importance of understanding these differences for unlocking the full potential of C. papaya leaves in medical and nutritional applications.