Resistance to most antimalarial drugs has spread from Southeast Asia to Africa. Accordingly, new therapies to use with artemisinin-based combination therapy (triple ACT) are urgently needed. Proveblue, a methylene blue preparation, was found to exhibit antimalarial activity against Plasmodium falciparum strains in vitro. Proveblue has synergistic effects when used in combination with dihydroartemisinin, and has been shown to significantly reduce or prevent cerebral malaria in mice. The objectives of the current study were to evaluate the in vitro baseline susceptibility of clinical field isolates to Proveblue, compare its activity with that of other standard antimalarial drugs and define the patterns of cross-susceptibility between Proveblue and conventional antimalarial drugs. The Proveblue IC50 of 76 P. falciparum isolates ranged from 0.5 nM to 135.1 nM, with a mean of 8.1 nM[ 95% confidence interval, 6.4-10.3]. Proveblue was found to be more active against P. falciparum parasites than chloroquine, quinine, monodesethylamodiaquine, mefloquine, piperaquine, doxycycline (P < 0.001) and lumefantrine (P = 0.014). Proveblue was as active as pyronaridine (P = 0.927), but was less active than dihydroartemisinin and artesunate (P < 0.001). The only significant cross-susceptibilities found were between Proveblue and dihydroartemisinin (r(2) = 0.195, P = 0.0001), artesunate (r(2) = 0.187, P = 0.0002) and piperaquine (r(2) = 0.063, P = 0.029). The present study clearly demonstrates the potential of Proveblue as an effective therapeutic agent against P. falciparum. In this context, the use of Proveblue as part of the triple ACT treatment for multidrug-resistant malaria warrants further investigation. (C) 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.