Two-dimensional diffusion-ordered NMR spectroscopy (DOSY) was used to examine a rotaxanated polymer and its blends with unthreaded macrocycles. The threaded macrocycles exhibit self-diffusion coefficients similar to those of the polymer, while the diffusion coefficients for the unthreaded macrocycles are much larger. For poly[(styrene)-rotaxa-(30-crown-10)], the method provided definitive proof that the 30-crown-10 is threaded; for blends, the method allowed quantitative determination of the threaded-macrocycle fraction. The DOSY technique is particularly useful for those rotaxanated polymers in which weakly interacting macrocycle and polymer offer no other detectable spectroscopic signature. Threading can be proven, and the fraction of threaded macrocycles can be determined.