In this report, our main focus is to introduce a set of one-dimensional (1D) NMR methods based on chemical shift, relaxation, and magnetization transfer, namely, NOE and chemical exchange involving selective pulse excitation to study the solution dynamics of drug in free and encapsulated state within polymeric microsphere. In this regard 5-fluorouracil (5-FU) loaded poly lactic-co-glycolic acid (PLGA) microspheres are prepared as model system via standard water-in-oil-in-water emulsification method. One-dimensional 1 H and 19 F nuclear magnetic resonance (NMR) spectra of 5-FU in presence of PLGA microspheres presented a significant change in linewidth and relaxation rates compared with free 5-FU confirming encapsulation. Furthermore, loss of coupling pattern in 1 H and 19 F NMR of PLGA encapsulated 5-FU as compared with free 5-FU suggests an enhanced –NH and –H 2 O protons exchange dynamics in the interior of the microsphere indicating hydrated microsphere cavity. Quantification of exchange dynamics in case of free and PLGA-encapsulated 5-FU was attempted employing 1D selective NOESY and 1D multiply selective inversion recovery experiments. Analysis of the exchange rates confirmed existence of more than one kind of water population within the cavity as mentioned in an earlier solid state NMR report. © 2018 John Wiley & Sons, Ltd.