Vitamin A deficiency remains prevalent in South Asia and sub-Saharan Africa. The well-established vitamin A supplementation program in children virtually reduces xerophthalmia and mortality in children but offers only a transient impact on raising serum retinol. Regular consumption of vitamin A-fortified food is considered a sustainable approach to maintain a healthy serum response of retinol of vulnerable populations. Bananas dominate the diet of many East-African countries where vitamin A deficiency is prevalent, thus making it a great vehicle for biofortification, a breeding technique to improve vitamin A value in the crop and increase dietary intake of vitamin A. Currently, provitamin A carotenoid (α- and β-carotene) biofortified bananas are being developed through genetic modification but its bioefficacy in humans has not been studied. The dominant provitamin A found in the biofortified bananas is α-carotene, followed by β-carotene. The nonsymmetrical α-carotene provides a molecule of active vitamin A (retinol) and its inactive analog (α-retinol). Exclusion of the inactive α-retinol with limited vitamin A activity is important for accurate determination of vitamin A activity of α-carotene in α-carotene containing foods such as provitamin A biofortified bananas. The overall objective of this study was to accurately determine the bioconversion factor of the α-carotene-containing provitamin A biofortified bananas in humans after the consumption of provitamin A biofortified bananas. An ultra-high selective and sensitive high-performance liquid chromatography–quadrupole-time-of-flight–high-resolution mass spectrometric with electrospray ionization in positive mode (HPLC-ESI (+)-QTOF-HRMS) quantitative method was developed and applied to the measurement of the postprandial appearance of provitamin A carotenoids and their retinyl ester bioconversion products as well as α-retinyl palmitate product of α-carotene after the consumption of provitamin A biofortified bananas. A 3µm C30 carotenoid column was used to separate α- and β-carotene, and their respective retinyl ester bioconversion derivative of α-retinol and retinol (i.e., α-retinyl ester and retinyl ester) in postprandial plasma triacylglycerol-rich lipoproteins. Labeled internal standards (d8-α-retinyl palmitate, 13C10-β-carotene, 13C10-retinyl palmitate) were used to account for analysis variability. Twelve healthy women each consumed three 200-g cooked banana fruit as follows: 1) biofortified banana fruit containing 1415.3 µg (2.64 µmol) of total β-carotene equivalents, 2) wild-type banana fruit with a β-carotene reference dose containing 536.8 μg (1.00 μmol) added β-carotene, and 3) wild-type banana fruit with a vitamin A reference dose containing 262.5 μg retinol activity equivalents (0.92 μmol) added retinyl palmitate. Mean (±SD) areas under the curve for retinyl palmitate in the TRL fractions (nmol·h) were 67.2 ± 50.5, 167.3 ± 114.5, and 167.8 ± 111.5 after consumption of the provitamin A-biofortified banana fruit, the wild-type banana fruit with the β-carotene reference dose, and the wild-type banana fruit with the vitamin A reference dose, respectively. The vitamin A equivalence of provitamin A of biofortified bananas and of wild-type banana fruit with the β-carotene reference dose were 18.52 ± 10.9 µg (mean ± SD) and 2.29 ± 0.93 µg, respectively. The vitamin A equivalency of provitamin A carotenoid biofortified banana fruit was similar to that reported for carotenoid-rich vegetables such as carrots or spinach, but less than that reported for starchy matrices (e.g., cassava, maize and rice).