Antisense oligonucleotides (ASOs) are emerging as a promising therapeutic approach for many disorders. Currently, fourteen ASOs have been FDA/EMA-approved, with many more in preclinical development. An important requirement for preclinical studies is the inclusion of negative controls to properly assess the efficacy of candidate ASOs. As commonly used scrambled or sense ASOs may elicit unwanted off-target effects that could influence downstream data analysis, there is a need for the inclusion of additional negative control ASOs that are well-characterized and well-tolerated. In this study, we designed and evaluated generic control ASOs (cASOs) across four different human cell lines and one murine fibroblast cell line. Characterization at the RNA and protein level revealed potential ASO toxicity mechanisms and species-specific responses as well as cell-type specific effects. In total, three cASOs with a distinct GC content were identified that do not exert off-target effects across different cell lines. To conclude, we report a set of cASOs that are potentially applicable in a broad variety of experiments using ASOs with similar physicochemical properties and across different cellular models.