Introduction. The in vitro assays for susceptibility of Plasmodium falciparum to antimalarial drugs are important tools for monitoring drug resistance, however few such studies have been undertaken in Colombia.Objective. P. falciparum isolates were obtained from several municipalities in western Colombia (Urabá, Bajo Cauca, Pacific Coast) and characterized for their in vivo susceptibility to chloroquine (CQ), amodiaquine (AQ), mefloquine (MQ), quinine (QN) and artesunate (AS).Material and methods. Patients with only P. falciparum infection (parasitemia=1,000 rings/µl) were included. Each antimalaria drug was tested with 7 dilutions, two-fold doses, with 2 replications and its effect evaluated using the histidine-rich protein (HRP-2) antigen detection method. Controls included FCB2 (chloroquine-resistant) and NF54 (chloroquine-sensitive) strains. IC50>100, 80, 64, 500 and 10.5 nM were used as the threshold criteria of resistance to CQ, AQ, MQ, QN and AS, respectively.Results. Twenty-five isolates were evaluated from Urabá (18), Bajo Cauca (2) and the Pacific Coast (5). The mean IC50 obtained with CQ, AQ, MQ, QN and AS were 422.9; 131.4; 56.3; 269.7 and 1.9 nM, respectively, and the number of resistant isolates for these drugs was 19 (76%), 4 (16%), 8 (32%), 6 (24%) and 1 (4%), respectively.Conclusions. The low sensitivity to CQ found here agrees with both in vitro and in vivo studies in Colombia. Ninety-six percent of the isolates were sensitive to AS. However, this study and previous reports have found isolates with low sensitivity to artemisinines (IC50>10.5 nM). This suggests that the indiscriminate use of these drugs put their efficacy at risk and eventually leave no options for falciparum malaria treatment.