Zika virus (ZIKV) and chikungunya virus (CHIKV) are important human pathogens and mosquito-borne arboviruses, which have resembling history, common vectors, circulating regions, and indistinguishable clinical symptoms. Wide geographical range that is suitable for ZIKV and CHIKV transmission underlines the concern about the impact of epidemic and endemic infections on burden of public health. In the present study, a highly sensitive and specific one-step multiplex real-time RT-PCR assay was developed and evaluated for simultaneous detection and quantification of ZIKV and CHIKV. The single reaction assay employs two pairs of primers and two TaqMan probes that differentiate ZIKV and CHIKV infections. The entire viral genomic RNA in vitro transcribed from full-length infectious clones were used to generate the standard curves for absolute quantification in subsequent tests. The detection limit of the one-step multiplex assay was 1 and 0.5 PFU for infectious ZIKV and CHIKV, respectively. The assessment of specificity indicated this assay is highly specific to targeted viruses showing no amplification of a variety of other flaviviruses. Our assay was able to detect geographically separated and phylogenetically diverse strains of ZIKV and CHIKV. On the applicability of monitoring viral multiplication in cells and testing clinical samples, the one-step multiplex assay provided efficient and accurate determination. The one-step multiplex real-time RT-PCR assay offers a valuable tool for detection of ZIKV and CHIKV and potentially contributes to general surveillance and clinical treatment.