For structural characterization from the primary to the 3D structure, direct fluorescent labeling, vaccine development, and numerous other studies, homogeneous and intact virus preparations of high purity are essential. Working with human rhinoviruses (HRVs), members of the picornavirus family and the main cause of generally mild respiratory infections, we noticed that our routine laboratory preparations of HRVs appeared highly homogeneous on analysis by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis, exclusively showing the four viral capsid proteins (VPs) which are only partly sequenced on the protein level. However, the preparations turned out to contain substantial amounts of contaminating material when analyzed by orthogonal analytical techniques including nano electrospray differential mobility analysis (nES DMA), capillary zone electrophoresis (UV fluorescence), and negative stain transmission electron microscopy. Because these latter analyses are not routine to many particularly biochemical/virology-oriented laboratories, the above contaminations might remain unnoticed and skew experimental and particularly functional results. By using HRV serotype A2 as example prepared by HeLa A1 cell culture and centrifugation we report monolithic anion-exchange chromatography as a last polishing step in the isolation/purification and demonstrate that it yields infective, highly pure, virus (was confirmed by peptide mass fingerprinting) devoid of foreign material as judged by the mentioned criteria. Exact size determination (29.5 nm) and the unsurpassed narrow width of the virus size distribution determined by nES DMA was achieved.