This review focuses on the recent advances on microscale separation technology, particularly on the new development on column and detection technologies in electrophoretic and chromatographic separation modes (i.e., CE, CEC, cLC, etc.). Representative examples illustrate also applications on the small molecules, peptides, proteins, polysaccharides, chiral and so on. 246 relevant articles are cited covering the literature published from 2013 to May 2017.