Many analytical methods employ self‐assembled nanostructured materials as chemical recognition media. Molecular permeation through these materials exhibits unique selectivity owing to nanoconfinement‐induced enhancement of permeant‐nanostructure interactions. This Personal Account introduces our efforts to investigate the detailed dynamics of single or a small number of molecules in nanostructured materials. We developed new experimental and analysis approaches built upon laser‐based fluorescence microscopy to measure the detailed translational and orientational dynamics of molecules diffusing in horizontally‐oriented, cylindrical nanostructures, including surfactant micelles, silica mesopores, block copolymer microdomains, and bolaamphiphile‐based organic nanotubes. Our studies clarified nanoscale details on the structural/chemical heterogeneity of the nanostructures, and their impacts on molecular mass transport dynamics.