Artemisinin-based combination therapies (ACTs) may select for malaria parasites with decreased drug sensitivity. We studied the sensitivity of parasites from children enrolled in treatment and prevention trials in Tororo, Uganda from June, 2010 to February, 2012. When Plasmodium falciparum malaria was diagnosed, blood was obtained, parasites (286 isolates) were cultured with serial dilutions of chloroquine (CQ), monodesethylamodiaquine (AQ), quinine (QN), dihydroartemisinin (DHA), lumefantrine (LM), or piperaquine (PQ) for 72 h, and ex vivo sensitivities were assessed by HRP-2-based ELISA. Sensitivities (nM) to CQ (median IC50 486.2; IQR 206.5-748.8), AQ (83.3; 58.4-132.4), PQ (20.3; 7.6-47.5) and QN (126.4; 74.9-196.3) varied widely; parasites were highly sensitive to LM (2.7; 0.97-6.7) and DHA (1.7; 1.0-2.8). IC50 values for 4 successive quartiles, each with 71-73 isolates collected over ~4 months, varied little for control strains (3D7 IC50s 0.54-1.8 nM) and all drugs except LM and PQ. For PQ, sensitivity decreased after the first quartile (median IC50 6.5), but was then stable (26.6-32.0). For LM, IC50s were low, but increased consistently (successive quartiles 1.1, 2.3, 3.5, and 6.2, p<0.05 for all comparisons except 2nd-3rd quartiles). Receiving monthly DHAPQ in a prevention trial was not associated with changes in PQ sensitivity compared to those receiving placebo. Having received artemether-LM (AL) within 60 days as treatment for a prior episode of malaria was associated with decreased sensitivity to LM (median IC50 1.59 for those without [n=84] vs. 3.24 for those with [n=119] recent AL; p=0.022 [GEE regression with log IC50 values]), but no significant change in sensitivity to the other study drugs. In summary, recent isolates of P. falciparum in Tororo were highly sensitive to components of AL, the national treatment regimen, but treatment with AL selected for parasites with decreased sensitivity, and overall sensitivities decreased from 2010 to 2012. Longitudinal surveillance of sensitivities of parasites under different selective drug pressures continues.