Live-attenuated intranasal vaccines offer great promise to generate mucosal immunity and reduce breakthrough infections after vaccination. Previously, we have developed a Newcastle disease virus (NDV)-based vaccine expressing the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), named NDV-HXP-S. This vaccine can be administered intranasally as a live-attenuated vector to provide both systemic and mucosal immunity. With the appearance of new SARS-CoV-2 variants of concern, new NDV-HXP-S variant-based vaccines have been developed. These new NDV-HXP-S variant vaccines can be combined in a multivalent formulation to extend their protection against other SARS-CoV-2 variants of concern. One of the main concerns when administering a live-attenuated vector intranasally is the possible vaccine shedding after vaccination. In the present work, we have developed a reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) method to evaluate NDV-HXP-S in vitro and in vivo along with an amplicon-based next-generation sequencing technique to quantify the relative percentage of each variant vaccine in the multivalent formulation. Both techniques were validated following the recommendations of the Food and Drug Administration and the European Medicines Agency. Finally, we applied both methods in in vitro cell culture experiments. The quantification of the NDV-based vaccines by RT-qPCR and the relative percentage of each variant vaccine were confirmed using a prototype trivalent formulation expressing the Ancestral (Wuhan), Beta, and Delta SARS-CoV-2 spikes. The present results will help the study of virus shedding in future clinical trials.Live-attenuated intranasal vaccines offer great promise to generate mucosal immunity and reduce breakthrough infections after vaccination. In this work, we have developed a combined reverse transcriptase-quantitative polymerase chain reaction and next-generation sequencing-directed approach to assess the viral shedding of multivalent Newcastle disease virus-based severe acute respiratory syndrome coronavirus 2 vaccines. This new formulation will be evaluated as a mucosal booster in phase I clinical trials next year.