21Background 22 The arsenal in anthelminthic treatment against schistosomiasis is limited and relies almost 23 exclusively on a single drug, praziquantel (PZQ). Thus, resistance to PZQ could constitute a 24 major threat. Even though PZQ is potent in killing adult worms, it has been shown to be limited 25 in its activity against earlier developmental stages. Current in vitro screening strategies for new 26 drugs depend on newly transformed schistosomulae (NTS) for initial hit identification, thereby 27 limiting sensitivity to new compounds predominantly active in later developmental stages. 28 Therefore, the aim of this study was to establish a highly standardized, straightforward and 29 reliable culture method to generate and maintain advanced larval stages in vitro. We present 30 here how this method can be a valuable tool to test drug efficacy at each discrete intermediate 31 larval stage, reducing the reliance on animal use (3Rs). 32Methodology/principal findings 33 Cercariae were mechanically transformed into skin-stage (SkS) schistosomulae and 34 successfully cultured under serum-free and cell-independent conditions for up to four weeks 35 with no loss in viability. Under these conditions, larval development halted at the lung-stage 36 (LuS). Addition of human serum (HSe) propelled further development into juvenile worms 37 within eight weeks. Skin and lung stages, as well as juvenile worms, were submitted to 96-well 38 format drug screening assays using known anti-schistosomal compounds such as PZQ, 39 oxamniquine (OXM), mefloquine (MFQ) and artemether (ART). Our findings showed stage-40 dependent differences in larval susceptibility. 41 Conclusion 42With this robust and highly standardized in vitro assay, important developmental stages of S. 43 mansoni up to juvenile worms can be generated and maintained over prolonged periods of time. 3 44The phenotype of juvenile worms when exposed to reference drugs was comparable to 45 previously published works for ex vivo harvested adult worms. Therefore, this in vitro assay 46 can help reduce reliance on animal experiments in the search for new anti-schistosomal drugs. 4 48 Author Summary 49 Schistosomiasis remains a major health threat, predominantly in developing countries. Even 50 though there has been some progress in search of new drugs, praziquantel remains the only 51 available drug. Probably the most important advance in the search for new drugs was in vitro 52 transformation of cercariae and their subsequent culture. However, hit identification in 53compound screenings is exclusively tested in skin stage parasites and is only confirmed for 54 more mature worms in a subsequent step. This is in part due to the lack of an easy culture system 55 for advanced-stage parasites. We present here a reliable and highly standardized way to 56 generate juvenile worms in vitro in a cell-free culture system. The inclusion of in vitro drug 57 tests on advanced-stage parasites in initial hit identification will help to identify compounds 58 that might otherwi...