Intra-patient tumor heterogeneity is likely a major determinant of clinical outcome in cancer patients. To assess heterogeneity in a minimally invasive manner, methods to perform single circulating tumor cell (CTC) genomics at high resolution are necessary. However, due to the rarity of CTCs, development of such methods is challenging. Here, we developed a modular single-CTC analysis pipeline to assess intra-patient heterogeneity by copy number (CN) profiling. To optimize this pipeline, spike-in experiments using MCF-7 breast cancer cells were performed. The VyCAP puncher system was used to isolate single cells. The quality of whole genome amplification (WGA) products generated by REPLI-g and Ampli1™ methods, as well as the results from the Illumina Truseq and the Ampli1™ LowPass library preparation techniques, were compared. Moreover, a bioinformatics pipeline was designed to generate CN profiles from single CTCs. The optimal combination of Ampli1™ WGA and Illumina Truseq library preparation was successfully validated on patient-derived CTCs. In conclusion, we developed a novel modular pipeline to isolate single CTCs and subsequently generate detailed patient-derived CN profiles that allow assessment of intra-patient heterogeneity in future studies.