Here, we advanced a novel technique using capillary ultrafiltration (CUF) probes to collect in vivo secreted proteins in the subcutaneous tissue of mouse ear. We fabricated two kinds of CUF probe, one with and one without a semi-permeable membrane hollow fiber. Proteins collected by CUF probes were profiled and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MADLI-TOF-MS) and quadrupole time-of-flight tandem mass spectrometry (Q-TOF-MS/MS) without using two-dimensional gel electrophoresis (2-DE) separation. Five proteins including cofilin-1, futuin-A, complement C3, gelsolin, and apolipoprotein C-1 were identified from the sample collected by the CUF probe with a semi-permeable membrane hollow fiber. The presence of well documented secretory proteins supports the efficiency of CUF probes in sampling in vivo secreted proteins. We also found that hemoglobin collected by the CUF probe without a semi-permeable membrane hollow fiber completely masked protein identification by mass spectrometry. The presence of relatively large amounts of hemoglobin in this condition illustrates the necessity of the semi-permeable membrane hollow fiber to the technique of CUF probe in conjunction with mass spectrometry. Also, the technique represents a powerful method for the identification of in vivo secreted proteins and has potential application for in the detection of biomarkers for human diseases. © 2005 Elsevier B.V. All rights reserved.