Summary: In the work presented here, a derivative of the heteroduplex tracking assay (HTA) which is capable of detecting multiple specific mutations in the HIV-1 protease gene is described. This multiple-site-specific (MSS) HTA is largely specific for mutations at codons 46, 48, 54, 82, 84, and 90, which have been implicated in resistance to commonly used protease inhibitors. It is demonstrated that MSS-HTA can reliably detect a 3% minority variant at high template numbers and that a 10% minority variant can be quantified reproducibly using as little as 1000 template molecules. Moreover, a generalized protocol for the generation of site-specific probes is introduced. MSS-HTA was used to investigate the natural history of ritonavir resistance evolution in vivo. It was shown that ritonavir can exert a strong selective pressure driving the emergence of novel pro variants for up to one year and that during this year multiple adaptive steps occur, each associated with a mean selective advantage of 0.18 and a mean number of coding mutations of 1.8, 1.3 of which occured at positions previously implicated in ritonavir resistance. Furthermore, it was demonstrated that strains with different inferred levels of resistance can coexist during the evolution of resistance. I proposed that this observation may be explained by oscillating drug concentrations that alternately favor resistant strains at high drug concentrations and susceptible strains at low drug concentrations. It was also demonstrated that decreased ritonavir susceptibility correlated with decreased infectivity and decreased processing of the NC-p1 protease cleavage site, suggesting that the NC-p1 processing defect may be the cause of the infectivity reduction. In an effort to determine the relationship of various assays of viral fitness, the results of several commonly used methods for fitness determination were compared for a set of nelfinavir resistant clones harboring the N88S mutation in protease. It was demonstrated that all four assays yield comparable results for this set of mutants using a mathematical framework developed in the appendix. Moreover, a reduction of the reverse transcriptase to capsid ratio was identified as another putative mechanism of fitness loss.