Since it was introduced in 1997, in-tube solid-phase microextraction (in-tube SPME), which uses a capillary column as extraction device, has been continuously developed as online microextraction coupled to LC systems (in-tube SPME-LC). In the last decade, new couplings have been evaluated on the basis of state-of-the-art LC instruments, including direct coupling of in-tube SPME to MS/MS systems, without chromatographic separation, for high-throughput analysis. In-tube SPME coupling to MS/MS has been possible thanks to the selectivity of capillary column coatings and MS/MS systems (SRM mode). Different types of capillary columns (wall-coated open-tubular, porous-layer open-tubular, sorbent-packed, porous monolithic rods, or fiber-packed) with selective stationary phases have been developed to increase the sorption capacity and selectivity of in-tube SPME. This review focuses on the in-tube SPME principle, extraction configurations, current advances in direct coupling to MS/MS systems, experimental parameters, coatings, and applications in different areas (food, biological, clinical, and environmental areas) over the last years.