Background: Anoikis is a form of programmed cell death triggered by the detachment of cells from the extracellular matrix. Anoikis resistance represents a critical factor in tumor metastasis, and elucidating the mechanisms by which epithelial cancer cells evade this process may provide a molecular insight for effectively targeting metastatic progression. Methods: Presented here are an experimental workflow and a detailed protocol to examine anoikis sensitivity in tumor cells both in vitro and in vivo. We described a detachment-induced anoikis model, a three-dimensional spheroid culture system, and an in vivo circulating tumor cell assay, by using the human lung carcinoma cell line A549 as a model system. We detailed the cell culture conditions, materials, and sample preparation, and the evaluation and quantification of anoikis. Together, these methods provide a comprehensive approach for investigating anoikis resistance. Conclusion: This protocol offers valuable insights into the mechanisms underlying anoikis resistance and may facilitate the identification of novel therapeutic targets for cancer treatment.