A sensitive and robust ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was established for the first time to simultaneously quantify marine neutral neoagaro-oligosaccharides (NAOS) and agar-oligosaccharides (AOS) with different degrees of polymerization (DP) in Hanks' balanced salt solution (HBSS). The separation was achieved on a BEH amide column using a mobile phase of acetonitrile-10 mmol L-1 ammonium acetate (58 : 42, v/v) with an isocratic elution program. The total analysis time was 3.5 min. The mass spectra were acquired in the multiple reaction monitoring (MRM) pattern by using a heated-electrospray ionization (H-ESI) source operating in the positive ionization mode. The linear range was 40-20 000 nmol L-1. The accuracy and precision ranged from 91.5 to 110.0% and 0.9 to 10.4%, respectively. The extraction recovery was consistent and reproducible. The stability was within 90.3-110.8%. The matrix effect, carryover, and dilution integrity were all satisfactory. Moreover, the validated method was successfully applied to the intestinal transport study by using the Caco-2 cell monolayer in vitro. The results revealed that neoagarobiose, neoagarotetraose, neoagarohexaose, agarotriose, agaropentose, and agaroheptose were transported by a paracellular pathway.