Protein separation through hollow fiber flow field-flow fractionation (HF FlFFF) at microflow rate regime was successfully achieved by employing a microbore hollow fiber. In most of the flow field-flow fractionation (FlFFF) techniques applied to the separation of proteins, including hollow fiber FlFFF (HF FlFFF), an outflow rate leading to a detector has typically been a few tenths of a milliliter per minute. In this study, it is demonstrated for the first time that 10 microL/min outflow rate in HF FlFFF can be employed for a successful separation of proteins by utilizing a small inner diameter (450 microm) hollow fiber. Initial evaluations of microbore HF FlFFF separation were made to improve separation efficiency by evaluating plate heights, sample recovery, and the limit of detection using protein standards. Microbore HF FlFFF was applied for the separation of low-abundance blood proteins depleted of high-abundance proteins from raw serum using immunoaffinity chromatography.