Methods for processing whole blood samples are provided. Aspects of the methods include depleting leukocytes from a whole blood sample to produce a leukocyte depleted sample, and then lysing red blood cells (RBCs) in the resultant leukocyte depleted sample to produce a leukocyte/RBC depleted sample. Also provided are compositions and kits for practicing embodiments of the invention. The methods and compositions find use in a variety of different applications, including the detection of circulating tumor cells (CTCs).