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Background The arsenal in anthelminthic treatment against schistosomiasis is limited and relies almost exclusively on a single drug, praziquantel (PZQ). Thus, resistance to PZQ could constitute a major threat. Even though PZQ is potent in killing adult worms, its activity against earlier stages is limited. Current in vitro drug screening strategies depend on newly transformed schistosomula (NTS) for initial hit identification, thereby limiting sensitivity to new compounds predominantly active in later developmental stages. Therefore, the aim of this study was to establish a highly standardized, straightforward and reliable culture method to generate and maintain advanced larval stages in vitro . We present here how this method can be a valuable tool to test drug efficacy at each intermediate larval stage, reducing the reliance on animal use (3Rs). Methodology/Principal findings Cercariae were mechanically transformed into skin-stage (SkS) schistosomula and successfully cultured for up to four weeks with no loss in viability in a commercially available medium. Under these serum- and cell-free conditions, development halted at the lung-stage (LuS). However, the addition of human serum (HSe) propelled further development into liver stage (LiS) worms within eight weeks. Skin and lung stages, as well as LiS, were submitted to 96-well drug screening assays using known anti-schistosomal compounds such as PZQ, oxamniquine (OXM), mefloquine (MFQ) and artemether (ART). Our findings showed stage-dependent differences in larval susceptibility to these compounds. Conclusion With this robust and highly standardized in vitro assay, important developmental stages of S . mansoni up to LiS worms can be generated and maintained over prolonged periods of time. The phenotype of LiS worms, when exposed to reference drugs, was comparable to most previously published works for ex vivo harvested adult worms. Therefore, this in vitro assay can help reduce reliance on animal experiments in search for new anti-schistosomal drugs.