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While antisense oligonucleotides (ASOs) are in principle capable of efficiently silencing gene expression, in silico prediction of the potency of ASO sequences is challenging. Identifying potent ASOs often requires an in vitro evaluation of many candidates that typically tile the target RNA of interest. To enable such analyses, we have developed a comprehensive and high-throughput platform to screen hundreds of ASOs for on-target knock-down efficiency. We established a panel of 20 model cell lines that have high free-uptake capacity and cover the majority of the human transcriptome. For each cell line, we established optimal seeding densities to maximize ASO delivery efficiency. An appropriate screening cell line with high expression of the target gene is selected based on available RNA-sequencing data. Candidate ASOs are delivered to 96-well culture plates in a single dose and target gene expression is quantified by RT-qPCR on crude cell lysates using 5 independent assays and multi-gene normalization. These data typically reveal one or multiple hotspot regions in the target gene that are highly accessible for ASO mediated knock-down. Based on these regions, additional ASOs can be designed for secondary screens in order to further expand the set of potent ASOs. Hits identified from these screens are further validated in dose response experiments to narrow down the list of candidates. Candidate ASOs are subsequently annotated based on their off-target gene potential through a proprietary in silico prediction pipeline considering the number of mismatches and their position in the ASO sequence. As a result, potent and specific ASOs can be identified for any target gene of interest. To demonstrate the robustness and potential applications of our workflow, we have screened over 10,000 ASOs with different chemistries targeting 250 genes in multiple cell lines. Our data reveal differences in the potency of ASO chemistries, varying targetability across genes, and differences in target gene knock-down among qPCR assays that need to be considered when screening for potent ASOs.