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Abstract: In general, analytical methods need various processes such as sampling, sample preparation, separation, detection and data analysis. Over 80% of analysis time is spent on the sampling and sample preparation steps such as extraction, concentration, fractionation and isolation of analytes. Therefore, it is not an exaggeration to say that choice of an appropriate sample preparation method greatly influences the reliable and accurate analysis. In-tube solid-phase microextraction (in-tube SPME) is a new effective sample preparation technique using a capillary column as an extraction device. Organic compounds in aqueous samples are directly extracted and concentrated into the stationary phase of a capillary column by repeated draw/eject cycles of sample solution, and they can be directly transferred to the liquid chromatographic system. In-tube SPME is an ideal sample preparation technique because of fast operation, easy to automation, solvent-free and inexpensive. On-line in-tube SPME can perform continuous extraction, concentration, desorption and injection using an autosampler, and is usually used in combination with high-performance liquid chromatography and liquid chromatography-mass spectrometry. This technique has successfully been applied to the determination of various compounds such as pesticides, drugs, environmental pollutants and food contaminants. In this review, an overview of the development of on-line in-tube SPME technique and its applications to forensic, clinical, environmental and food analyses are presented. New approaches of in-tube SPME technique using a fiber packed capillary, monolith capillary, polypyrrole coated capillary and molecularly imprinted capillary are also described