Virus-like particles (VLPs) have been extensively explored as vaccine candidates since the mid-1980s. Numerous VLPs have been designed as vaccines for prevention of virus-induced infectious diseases and for the therapeutical treatment of chronic diseases and drug addiction. Recently, a vaccine against nicotine addiction, which is based on VLPs of the RNA phage Qb to which nicotine haptens are covalently coupled via succinimate linkers (NicQb), has attracted a great deal of interest. Phase II clinical trials with this vaccine have shown that it is efficacious for smoking cessation in humans when antinicotine antibody levels are sufficiently high. For commercialization, the development of stable formulations enabling storage for prolonged periods is required. Hereby, lyophilization, a well-established method leading to stable and dry formulations, is often applied. In this study, we investigated the influence of different pH values and various excipients such as surfactants, polyols, sugars, and salts on the stability of NicQb in liquid formulations, during freeze thawing, freeze drying, and finally upon storage of the dried product. Lyophilized NicQb formulations were developed which were stable over 6 months at ambient temperature with fully retained biological activity. Hereby, it was found that a combination of the surfactant polysorbate 20 and the disaccharide trehalose was capable to prevent NicQb aggregation and to preserve its integrity (nicotine binding and integrity of VLP shell). Furthermore, asymmetrical flow field-flow fractionation (AF4), a new, promising analytical tool, was established for the investigation of VLP stability.