This review highlights on-line sample preconcentration by large-volume sample stacking with an electroosmotic flow pump (LVSEP) in microscale electrophoresis. LVSEP requires only a hydrodynamic sample injection into the whole capillary/microchannel and a constant voltage application, i.e., without polarity switching, providing an efficient sensitivity enhancement with a relatively easy experimental procedure without loss of the separation efficiencies. Recent developments and applications of LVSEP are briefly reviewed from the first LVSEP study in 1999 with categorization on the basis of separation modes, including zone electrophoresis, cyclodextrin- and affinity-based separation modes and non-aqueous capillary electrophoresis. © The Japan Society for Analytical Chemistry.