The present study focuses on development of phytochemical methods for quality assessment of two West-African Cochlospermum species (Cochlospermum planchonii and Cochlospermum tinctorium) traditionally used for malaria treatment in Burkina Faso. Antimalarial activity of preparations from dried rhizomes (decoction) was tested against the chloroquine-sensitive Plasmodium strain 3D7 using the histidine-rich protein II (HRP2) drug susceptibility assay and compared with extract preparations using organic solvents of different polarity. Two main apocarotenoids were isolated from rhizomes of C. planchonii and unambiguously identified as dihydrocochloxanthine and cochloxanthine by spectroscopic methods. Comparative HPLC analyses of thirty-nine (39) samples from markets and from collections in natural habitats of both species showed a high variability in the accumulation of cochloxanthines and related carotenoids which were proven to be characteristic for rhizomes of both species and generally absent in leaves. Furthermore, content of total phenolics and antioxidant activities (DPPH and FRAP) as well as haemolytic activity of various extracts was tested. The HPLC method presented here was validated and provides a good separation of both compounds including 10 minor carotenoids. Extracts from both species and pure cochloxanthine offered pronounced antioxidant activities and weak haemolytic activity while, in contrast, dihydrocochloxanthine had a strong haemolytic effect at the highest concentration analysed. However, cochloxanthine as well as dihydrocochloxanthine showed erythroprotective effects against the haemolytic activity of the reference saponin. Moderate antiplasmodial activity between 16 and 63 μg/ml were observed with all tested extracts, and lower IC50 values were obtained with pure dihydrocochloxanthine (IC50=6.9 μg/ml), cochloxanthine (IC50=6.8 μg/ml), the DCM fraction (IC50=2.4 μg/ml) and the ethyl acetate fraction (IC50=11.5μg/ml) derived from a methanolic extract of C. planchonii. This study shows a major variability of carotenoid content and antiplasmodial activity of both C. planchonii and C. tinctorium. The high haemolytic activity of dihydrocochloxanthine (at 100 μg/ml) should be considered as a selection criterion for choosing species phenotypes for treatment.