The structural and functional heterogeneity of the collecting duct presents a tremendous experimental challenge requiring manual microdissection, which is time consuming, labor intensive, and not amenable to high throughput. To overcome these limitations we developed a novel approach combining the use of transgenic mice expressing green fluorescent protein (GFP) in the collecting duct with large particle based flow cytometry to isolate pure populations of tubular fragments from the whole collecting duct (CD), or inner medullary (IMCD), outer medullary (OMCD) or connecting segment/cortical collecting duct (CNT/CCD). Kidneys were enzymatically dispersed into tubular fragments and sorted based on tubular length and GFP intensity using large particle based flow cytometry or Complex Object Parametric Analyzer Sorter (COPAS^TM). A LIVE/DEAD assay demonstrates that the tubules were greater than 90% viable. Tubules were collected as a function of fluorescent intensity and analyzed by epifluorescence and phase microscopy for count accuracy, GFP positivity, average tubule length, and time required to collect 100 tubules. Likewise, mRNA and protein from sorted tubules was analyzed for expression of tubule segment-specific genes using quantitative real-time RT-PCR and immunoblotting. The purity and yield of sorted tubules was related to sort stringency. Four to six replicates of 100 collecting ducts (9.68 ± 0.44 to 14.5 ± 0.66 cm or 9.2 ± 0.7 mg tubular protein) were routinely obtained from a single mouse in under an hour. In conclusion, large particle based flow cytometry is fast, reproducible, and generates sufficient amounts of highly pure and viable collecting ducts from single or replicate animals for gene expression and proteomic analysis.