Circulating tumor cells (CTC) are rare cells which have left the primary tumor to enter the blood stream. Although only a small CTC subgroup is capable of extravasating, the presence of CTCs is associated with an increased risk of metastasis and a shorter overall survival. Understanding the heterogeneous CTC biology will optimize treatment decisions and will thereby improve patient outcome. For this, robust workflows for detection and isolation of CTCs are urgently required. Here, we present a workflow to characterize CTCs by combining the advantages of both the CellSearch((R)) and the CellCelector micromanipulation system. CTCs were isolated from CellSearch((R)) cartridges using the CellCelector system and were deposited into PCR tubes for subsequent molecular analysis (whole genome amplification (WGA) and massive parallel multigene sequencing). By a CellCelector screen we reidentified 97% of CellSearch((R)) SKBR-3 cells. Furthermore, we isolated 97% of CellSearch((R))-proven patient CTCs using the CellCelector system. Therein, we found an almost perfect correlation of R-2=0.98 (Spearman's rho correlation, n=20, p<0.00001) between the CellSearch((R)) CTC count (n=271) and the CellCelector detected CTCs (n=252). Isolated CTCs were analyzed by WGA and massive parallel multigene sequencing. In total, single nucleotide polymorphisms (SNPs) could be detected in 50 genes in seven CTCs, 12 MCF-7, and 3 T47D cells, respectively. Taken together, CTC quantification via the CellCelector system ensures a comprehensive detection of CTCs preidentified by the CellSearch((R)) system. Moreover, the isolation of CTCs after CellSearch((R)) using the CellCelector system guarantees for CTC enrichment without any contaminants enabling subsequent high throughput genomic analyses on single cell level. (c) 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:125-132, 2017