•Nine antiviral compounds were evaluated against chikungunya, dengue and Junin viruses using different dye indicators.•AlamarBlue, neutral red, Viral ToxGlo and WST-1 dyes performed similarly in quantifying antiviral effects.•Alamar Blue underestimated the cytotoxicity of some of the test compounds compared to the other dyes.•Viral ToxGlo uptake into dengue-infected cells was nearly twice that of other dyes and data generated were more variable.•Certain dyes could be used sequentially in the same cell cultures. Studies were conducted to determine the performance of four dyes in assessing antiviral activities of compounds against three RNA viruses with differing cytopathogenic properties. Dyes included alamarBlue® measured by absorbance (ALB-A) and fluorescence (ALB-F), neutral red (NR), Viral ToxGlo™ (VTG), and WST-1. Viruses were chikungunya, dengue type 2, and Junin, which generally cause 100, 80–90, and 50% maximal cytopathic effect (CPE), respectively, in Vero or Vero 76 cells Compounds evaluated were 6-azauridine, BCX-4430, 3-deazaguanine, EICAR, favipiravir, infergen, mycophenolic acid (MPA), ribavirin, and tiazofurin. The 50% virus-inhibitory (EC50) values for each inhibitor and virus combination did not vary significantly based on the dye used. However, dyes varied in distinguishing the vitality of virus-infected cultures when not all cells were killed by virus infection. For example, VTG uptake into dengue-infected cells was nearly 50% when visual examination showed only 10–20% cell survival. ALB-A measured infected cell viability differently than ALB-F as follows: 16% versus 32% (dengue-infected), respectively, and 51% versus 72% (Junin-infected), respectively. Cytotoxicity (CC50) assays with dyes in uninfected proliferating cells produced similar CC50 values for EICAR (1.5–8.9μM) and MPA (0.8–2.5μM). 6-Azauridine toxicity was 6.1–17.5μM with NR, VTG, and WST-1, compared to 48–92μM with ALB-A and ALB-F (P<0.001). Curiously, the CC50 values for 3-deazaguanine were 83–93μM with ALB-F versus 2.4–7.0μM with all other dyes including ALB-A (P<0.001). Overall, ALB minimized the toxicities detected with these two inhibitors. Because the choice of dyes affected CC50 values, this impacted on the resulting in vitro selectivity indexes (calculated as CC50/EC50 ratio).