Mutations in the human immunodeficiency virus (HIV) enable virus replication even when appropriate antiretroviral therapy is followed, thus leading to the emergence of drug resistance. In a previous work, we systematically examined seven single mutations that are associated with saquinavir (SQV) resistance in HIV-1 protease (Tzoupis, H.; Leonis, G.; Mavromoustakos, T.; Papadopoulos, M. G. J. Chem. Theory Comput. 2013, 9, 1754-1764). Herein, we extend our analysis, which includes seven double (G48V-V82A, L10I-G48V, G48V-L90M, I84V-L90M, L10I-V82A, L10I-L63P, A71V-G73S) and four multiple (L10I-L63P-A71V, L10I-G48V-V82A, G73S-I84V-L90M, L10I-L63P-A71V-G73S-I84V-L90M) SQV HIV-1 PR mutant complexes, in an attempt to generalize our findings and formulate the main elements of the SQV resistance mechanism in the protease. On the basis of molecular dynamics (MD), molecular mechanics Poisson Boltzmann surface area (MM PBSA), and ab initio computational approaches, we identified specific features that constitute the HIV-1 PR mechanism of resistance at the molecular level: the low flexibility of SQV in the binding cavity and the preservation of hydrogen bonding (HB) and van der Waals interactions between SQV and several active-site (Gly27/27', Asp29/29'130/30', especially Asp25/25') and flap (Ile50/50', Gly48/48') residues of the protease contribute significantly to efficient binding. The total enthalpy loss in all mutants is mostly due to the loss in enthalpy of the active-site region. Furthermore, it was observed that mutation accumulation may induce stabilization to SQV and to the flaps through enhanced HE interactions that lead to improved inhibition (e.g., accumulation of mutations in complexes containing L10I, G48V, L63P, I84V, or L90M single mutations). It was also concluded that permanent flap closure is obtained independently of mutations and SQV binding is mostly driven by van der Waals, nonpolar, and exchangeenergy contributions. Importantly, it was indicated that the optimal positioning of SQV and the structure of the binding cavity are tightly coupled, since small changes in geometry may affect the binding energy greatly. The results of our theoretical approaches are in agreement with experimental evidence and provide a reliable description of SQV resistance in HIV-1 PR.