Background: Biomarkers to assess early response to cisplatin based neoadjuvant chemotherapy (NAC) in MIBC is a clinical need. We hypothesized that CTC enumeration and genomic profiling using a novel microfluidic immunomagnetic assay IsoFlux (Fluxion Biosciences Inc., CA) could provide novel biomarkers in individual patients during NAC. Methods: Peripheral blood was drawn at baseline and after one cycle (3-4 weeks) of NAC in patients (pts) with locally advanced resectable bladder cancer in an IRB approved prospective study at the University of Michigan. CTCs were enumerated by the CellSearch and IsoFlux assays in parallel and by IsoFlux in duplicate samples. We correlated CTC numbers with pStage at cystectomy, a known prognostic variable for survival. We assessed gene expression by RT-PCR and gene mutational status by Ion Torrent sequencing on CTCs. Results: We analyzed 21 pts with localized muscle invasive bladder cancer (cT2-T4aNx). Mean age was 65 yrs with 84% male. Clinical stage was 71% cT2N0, 14% cT3N0, 10% cT4N0, 5% cT4aN1. NAC was GC in 9 pts and MVAC in 12 pts. The IsoFlux assay detected more CTCs per sample than CellSearch at baseline (median =10 vs. 0, n=5) and after 1 cycle (5.5 vs. 0, n=4). IsoFlux assay in parallel tubes had 80% of samples with <5 CTC difference between the pairs. Baseline CTC concordance with pStage was 75% (12/16) (concordance=either <pT1N0 with <10 CTCs or ≥ pT1N0 with ≥10 CTCs). On the other hand, concordance of cStage with pStage was only 62.5% (10/16). PDGFRA, PIK3CA and MET mutations were detected on CTCs. Interrogation of CTCs for gene expression by RT-PCR and application of next generation sequencing (NGS) to CTCs were feasible. Conclusions: IsoFlux detects more CTCs than CellSearch in patients undergoing NAC. Baseline CTC number is a potential prognostic variable superior to cStage. However, changes in CTC number before and after 1 NAC cycle do not appear to reflect benefit in individual patients. Gene expression and genomic profiling of CTCs are feasible. CTCs are potential liquid biopsies in patients undergoing NAC in bladder cancer.