This paper presents a novel splitting method for liquid chromatography/mass spectrometry (LC/MS) application, which allows fast MS detection of LC-separated analytes and subsequent online analyte collection. In this approach, a PEEK capillary tube with a micro-orifice drilled on the tube side wall is used to connect with LC column. A small portion of LC eluent emerging from the orifice can be directly ionized by desorption electrospray ionization (DESI) with negligible time delay (6~10 ms) while the remaining analytes exiting the tube outlet can be collected. The DESI-MS analysis of eluted compounds shows narrow peaks and high sensitivity because of the extremely small dead volume of the orifice used for LC eluent splitting (as low as 4 nL) and the freedom to choose favorable DESI spray solvent. In addition, online derivatization using reactive DESI is possible for supercharging proteins and for enhancing their signals without introducing extra dead volume. Unlike UV detector used in traditional preparative LC experiments, this method is applicable to compounds without chromophores (e.g., saccharides) due to the use of MS detector. Furthermore, this splitting method well suits monolithic column-based ultra-fast LC separation at a high elution flow rate of 4 mL/min. Figure ᅟ