A panel of 245 clinical samples with known treatment histories was retrospectively evaluated for cross-resistance to new protease inhibitors (PI). Samples with resistance to previously approved PI displayed high cross-resistance to atazanavir, whereas cross-resistance to amprenavir was considerably lower. A similar cross-resistance profile was observed for lopinavir, if a higher cut-off for resistance (9.5-fold) was applied. The enhanced efficacy of boosted PI is discussed with respect to clinically relevant cut-offs for drug resistance.