Porous-wall hollow glass microspheres (PWHGMs) are a form of glass materials that consist of 1-μm-thick porous silica shells, 20-100 μm in diameter, with a hollow cavity in the center. Utilizing the central cavity for material storage and the porous walls for controlled release is a unique combination that renders PWHGMs a superior vehicle for targeted drug delivery. In this study, loaded PWHGMs were characterized for the first time employing proton nuclear magnetic resonance (1H NMR) spectroscopy. A vacuum-based loading system was developed to load PWHGMs with various liquid materials followed by a washing procedure that uses solvents immiscible with the loaded materials. Immiscible binary model systems (chloroform/water, n-dodecane/water), as well as the hydrolysis reaction of isopropyl acetate, were investigated to obtain 1H NMR evidence of loading materials into PWHGMs and their subsequent release to the surrounding solutions. The unique 1H NMR peak shapes and relative integrals of the materials loaded in PWHGMs were distinguishable from those of the same materials in the surrounding solutions. Encounters of isopropyl acetate contained in the PWHGMs with acid protons from concentrated H2SO4 added to the surrounding solution become evident by the formation of the reaction product isopropanol. PWHGMs loaded with H2O and suspended in D2O were used to obtain quantitative release kinetics of H2O-loaded PWHGMs. A five-parameter double-exponential curve fit of experimental 1H NMR signal intensities as a function of time indicated two release rates for H2O from H2O-loaded PWHGMs suspended in D2O with one time constant of 18-20 min and another one of 160 min. The two disparate release-rate time constants are consistent with loaded PWHGMs with breached and unbreached porous walls. The results demonstrate that 1H NMR spectroscopy is particularly useful for investigating formulations and applications of PWHGMs in targeted drug delivery.