Factors affecting relative fitness measurements in pairwise competition assays of human immunodeficiency viruses.

•Mutations in human immunodeficiency virus type 1 (HIV-1) confer drug resistance and immunological escape.•Cell culture growth competition assays are used to estimate viral fitness and quantify the impact of these mutations.•We optimized a set of parameters with which to assess small differences in the relative fitness of HIV-1 variants in pairwise competition assays.•The optimized protocol uses: (a) viral growth determined from monoinfection experiments; (b) MOI of 0.005 in competition assays; (c) consistent ratio of parental and mutant viruses in competition assays; (d) the multipoint 1+s algorithm for assessing fitness differences within the logarithmic phase of viral growth.