The Chikungunya virus (CHIKV) belongs to the Alphavirus genus of Togaviridae family and contains a positive-sense single stranded RNA genome. Infection by this virus mainly causes sudden high fever, rashes, headache, and severe joint pain that can last for several months or years. CHIKV, a mosquito-borne arbovirus, is considered a re-emerging pathogen that has become one of the most pressing global health concerns due to a rapid increase in epidemics. Because handling of CHIKV is restricted to Biosafety Level 3 (BSL-3) facilities, the evaluation of prophylactic vaccines or antivirals has been substantially hampered. In this study, we first iden-tified the whole structural polyprotein sequence of a CHIKV strain isolated in South Korea (KNIH/2009/77). Phylogenetic analysis showed that this sequence clustered within the East/ Central/South African CHIKV genotype. Using this sequence information, we constructed a CHIKV-pseudotyped lenti-virus expressing the structural polyprotein of the Korean CHIKV isolate (CHIKVpseudo) and dual reporter genes of green fluorescence protein and luciferase. We then developed a pseudovirus-based neutralization assay (PBNA) using CHIKVpseudo. Results from this assay compared to those from the conventional plaque reduction neutralization test showed that our PBNA was a reliable and rapid method to evaluate the efficacy of neutralizing antibodies. More importantly, the neutralizing activities of human sera from CHIKV-infected individuals were quantitated by PBNA using CHIKVpseudo. Taken together, these results suggest that our PBNA for CHIKV may serve as a useful and safe method for testing the neutralizing activity of antibodies against CHIKV in BSL-2 facilities.