We developed an epithelial cell adhesion molecule (EpCAM)-based positive method and CD45/CD66b-based negative method for isolating circulating tumor cells (CTCs) by lateral magnetophoresis. The CTC recovery rate, white blood cell depletion rate, and purity of CTCs isolated using the positive and negative methods were analyzed using blood samples spiked with cancer cells with different expression levels of EpCAM. The aim was to assess the strengths and weaknesses of the positive and negative isolation methods for CTC-based diagnostics, prognostics, and therapeutics for cancer. The EpCAM-based positive method yielded CTCs of high purity, while the CD45/CD66b-based negative method yielded a large number of CTCs. In conclusion, the positive method shows promise for detecting somatic oncogenic mutations and the negative method shows promise for discovery of cellular and transcriptomic biomarkers of cancer.