Virus-like particles have been successfully used as safe vaccines, as their structure is identical to their native counterparts but devoid of the viral genetic material. However, production of these complex structures is not easy, as recombinant proteins must assemble into virus-like particles. Techniques to differentiate assembled and soluble proteins, as well as assembly intermediaries often present in a sample, are required. An example of complex virus-like particles mixture occurs when rotavirus proteins are recombinantly expressed. Rotavirus-like particles (RLP) can be single (sl), double (dl), or triple layered (tl). The use of RLP preparations as vaccines requires their complete characterization, including separation and quantification of each RLP in a sample. In this work, CZE was evaluated for the separation and quantification of dl and triple-layered rotavirus-like particles (t1RLP). A fused-silica capillary with a deoxycholate running buffer efficiently separated dl and t1RLP in RLP preparations, as they migrated in two discrete peaks with electrophoretic mobilities of 1.24 +/- 0.04 and 2.95 +/- 0.03 Ti, respectively. Standard curves for dl and t1RLP were generated, and the response was linearly proportional to analyte concentration. The methodology developed was quantitative, specific, accurate, precise, and reproducible. CZE allowed the quantitative characterization of RLP preparations, which is required for evaluation of immunogens, for process development, and for quality control protocols.