Sequential-injection analysis (SIA) is a sample handling and manipulation technique developed to address some limitations of traditional on-line analysis approaches, mainly flow-injection analysis (FIA). The heart of a SIA manifold is a multi-position selection valve. The common port of this valve is connected to a so-called “holding coil” (essentially a long piece of coiled tubing) while the selection ports of the valve can be connected to various flow-through modules. The sample to be analyzed is initially aspirated and stored in the holding coil and can be further serially processed by directing it to appropriate modules in which both physical manipulation and/or chemical transformations may take place. SIA has found numerous applications in the field of clinical and bio-analytical chemistry by exploiting its potential as a sample-handling and manipulation technique. This review will cover the most significant applications of SIA in this area including on-line dilution/mixing, liquid and solid-phase extraction and pre-concentration, immunoassays, enzymatic methods, coupling to separation modules, miniaturization and other approaches for the determination of compounds with biological and clinical significance.