As important driving forces for building a wide variety of architectures and assemblies in macroscopic systems, non-covalent interactions are individually weak but collectively important. For decades of on-going pursuits on the physical origins, the portfolio of non-covalent interactions has largely expanded from conventional hydrogen bonds to diverse chemical combinations with different structural and energetic boundaries. Among many experimental techniques, rotational spectroscopy can not only offer unexplored avenues for high resolution studies in gas-phase, but also unravel the nature of non-covalent interactions in condensed phases, avoiding the interference of environmental factors. In addition, rotational spectroscopy is arguably the most accurate molecular spectroscopic technique due to its high sensitivity to mass distributions of isolated molecules and molecular complexes, even subtle differences in mass distribution (arising from isotopic substitution, isomerization, tautomerization or conformerization) can lead significant changes in the pattern of rotational transitions. In this review, the basic principles and advantages of rotational spectroscopy in characterizing non-covalent interactions are briefly introduced firstly. Then, the lastest achievements of rotational investigations in σ-hole and π-hole non-covalent interactions are comprehensively reviewed, which fully shows the ability of rotational spectroscopy in structural and energetic assessment of inter molecular non-covalent interactions, and indicates its potential contribution to the transition from fundamental understandings to applications in supramolecular chemistry and crystal engineering.