Analyzing the chemical compounds in biological fluid, such as human blood, by mass spectrometry, sample pretreatments are frequently needed. Those methods, including centrifugation, solid phase extraction (SPE) and liquid-liquid extraction, separate and remove the interferences in biological samples prior to mass analysis. The drawbacks of sample pretreatments mentioned above are time consuming and tedious operation procedures. Thin layer chromatography (TLC) is an analytical tool commonly used for sample purification and separation. To characterize the chemical compounds on TLC plate, coupling TLC with mass spectrometry seems to be a most efficient approach. Since the analytes are buried in the silica layer of TLC plate, it is important to develop a sampling method to transfer analytes from silica layer to mass analyzer. We develop a technique named ¡§TLCspray¡¨ to analyzing the chemical compounds deposited on TLC plate. The concept of TLCspray is similar to the direct electrospray probe (DEP) which has been described by our group: droplets deposed on a metal coated rod can producing ESI plume while applying high voltage. The TLC plate used for TLCspray is cut into stripes with sharp end. Samples are applied on the TLC stripes, than mount onto a holder which fix TLC stripes in front of mass inlet. The mobile phase flow through sample spot, and carry the analyte to the edge of TLC stripe where the fine ESI plume is produced in the effect of electric field. Complex matrixes such as red blood cell (RBC) and proteins are stop on the original position by the silica layer which acts as filter to clean up the unwanted interferences in biological samples. Zolpidem, a drug used to treat insomnia, is chose to demonstrate the sensitivity of TLCspray. The LOQ of zolpidem is 50 ng/mL, and linear dynamic range is 50-1000 ng/mL in human blood. The analysis process of TLCspray for human blood can be finished within 4 minutes. Comparing to other sample pretreatment prior to mass analysis for biological samples, TLCspray can reduce the time needed for clean up the samples without loss the sensitivity.