We investigated the stage-specific mechanisms of partial resistance to artemisinin (ART, an antimalarial drug) in Plasmodium falciparum (P. falciparum) carrying the Kelch13 C580Y mutation.Using fluorescence labeling and activity-based protein profiling, we systematically profile the ART activation levels in P. falciparum during the entire intra-erythrocytic developmental cycle (IDC), and determined the ART-targets profile of the ART-sensitive and -resistant strains at different stages. We retrieved and integrated datasets of single-cell transcriptomics and label-free proteomics across three IDC stages of wild-type P. falciparum. We also employed lipidomics to validate lipid metabolic reprogramming in the resistant strain.The activation and expression patterns of genes and proteins of ART-targets in both ART-sensitive and resistant strains varied at different stages and periods of P. falciparum development, with the late trophozoite stage harboring the largest number of ART targets. We identified and validated 36 overlapping targets, such as GAPDH, EGF-1a, and SpdSyn, during the IDC stages in both strains. We revealed the ART-insensitivity of fatty acid-associated activities in the partially resistant strain at both the early ring and early trophozoite stages.Our multi-omics strategies provide novel insights into the mechanisms of ART partial resistance in Kelch13 mutant P. falciparum, demonstrating the stage-specific interaction between ART and malaria parasites.Copyright © 2023 Elsevier Ltd. All rights reserved.