Insight into the mode of action of newly discovered antiviral agents is now almost a prerequisite for clinical development. This protocol describes a method that provides information on the target of inhibitors of the human immunodeficiency virus (HIV); it can also be adapted to other viruses. The results from this experiment are available within 2 d. This time-based approach determines how long the addition of a compound can be postponed before losing its antiviral activity in cell culture. The target of an antiviral compound can be identified by comparing its relative position in the time scale to that of reference drugs. Therefore, it is more precise than, for example, in the case of HIV, a determination of pre- or postintegrational mode of action, and combines in one routine different assays for studying mechanisms of action.