Nucleic acid-based PS-ASOs have the potential to activate cellular innate immune responses, and the level of activation can vary quite dramatically with sequence. Minimizing degree of proinflammatory effect is one of the main selection criteria for compounds intended to move into clinical trials. While a recently developed hPBMC-based assay showed excellent ability to detect innate immune active PS-ASOs, which can then be discarded from the developmental process, this assay is highly resource-intensive and easily affected by subject variability. This compelled us to develop of a more convenient high-throughput assay. Here, we describe a new in vitro assay, utilizing a cultured human Bjab cell line, which was developed and validated to identify PS-ASOs that may cause innate immune activation. The assay was calibrated to replicate results from the hPMBC assay. The Bjab assay was designed to be high throughput and more convenient by using RT-qPCR readout of mRNA of the chemokine Ccl22. The Bjab assay was also shown to be highly reproducible and to provide a large dynamic range in determining the immune potential of PS-ASOs via comparison to known benchmark PS-ASO controls that were previously shown to be either safe or inflammatory in clinical trials. In addition, we demonstrate that Bjab cells can be used to provide mechanistic information on PS-ASO-TLR9 dependent innate immune activation.