Hepatitis C virus (HCV) infection is the leading cause of liver transplantation in Western countries. Studies of HCV infection using cell culture-produced HCV (HCVcc) in vitro systems require quantification of infectious HCV virions, which has conventionally been performed by immunofluorescence-based focus-forming assay with manual foci counting; however, this is a laborious and time-consuming procedure with potentially biased results. In the present study, we established and optimized a method for convenient and objective quantification of HCV virions by colorimetric focus-forming assay with automated focus counting by image analysis. In testing different enzymes and chromogenic substrates, we obtained superior foci development using alkaline phosphatase-conjugated secondary antibody with BCIP/NBT chromogenic substrate. We additionally found that type I collagen coating minimized cell detachment during vigorous washing of the assay plate. After the colorimetric focus-forming assay, the foci number was determined using an ELISpot reader and image analysis software. The foci number and the calculated viral titer determined by this method strongly correlated with those determined by immunofluorescence-based focus-forming assay and manual foci counting. These results indicate that colorimetric focus-forming assay with automated focus counting by image analysis is applicable as a more-efficient and objective method for quantification of infectious HCV virions.