The rapid development of antimalarial resistance motivates the continued search for novel compounds with a mode of action (MoA) different to current antimalarials. Phenotypic screening has delivered thousands of promising hit compounds without prior knowledge of the compounds’ exact target or MoA. Whilst the latter is not initially required to progress a compound in a medicinal chemistry program, identifying the MoA early can accelerate hit prioritization, hit-to-lead optimization and preclinical combination studies in malaria research. The effects of drug treatment on a cell can be observed on systems level in changes in the transcriptome, proteome and metabolome. Machine learning (ML) algorithms are powerful tools able to deconvolute such complex chemically-induced transcriptional signatures to identify pathways on which a compound act and in this manner provide an indication of the MoA of a compound. In this study, we assessed different ML approaches for their ability to stratify antimalarial compounds based on varied chemically-induced transcriptional responses. We developed a rational gene selection approach that could identify predictive features for MoA to train and generate ML models. The best performing model could stratify compounds with similar MoA with a classification accuracy of 76.6 ± 6.4%. Moreover, only a limited set of 50 biomarkers was required to stratify compounds with similar MoA and define chemo-transcriptomic fingerprints for each compound. These fingerprints were unique for each compound and compounds with similar targets/MoA clustered together. The ML model was specific and sensitive enough to group new compounds into MoAs associated with their predicted target and was robust enough to be extended to also generate chemo-transcriptomic fingerprints for additional life cycle stages like immature gametocytes. This work therefore contributes a new strategy to rapidly, specifically and sensitively indicate the MoA of compounds based on chemo-transcriptomic fingerprints and holds promise to accelerate antimalarial drug discovery programs.