The strategy developed in this report consisted of synthesizing polystyrene (PS) blocks by nitroxide-mediated controlled radical polymerization from poly(ethylene oxide) (PEO) macroinitiators. Difunctional macroinitiators were first prepared in a two-step procedure by reacting PEO (Mn ranging from 1450 to 35000 g/mol) with MONAMS, an alkoxyamine based on N-tert-butyl-N-(1-diethylphosphono-2,2-dimethylpropyl)nitroxide, called SG1. A series of polystyrene-block-poly(ethylene oxide)-block-polystyrene (PS-b-PEO-b-PS) triblock copolymers was then synthesized with various chain dimensions and PEO block mass fractions from 0.13 to 0.65. Precise insight into the prepared triblock copolymer structure in terms of molecular weight, block length, and polydispersity index was obtained using complementary characterization techniques such as 1H NMR spectroscopy and size exclusion chromatography (SEC). These data were completed by two-dimensional chromatography, a combination of liquid chromatography at the critical conditions and SEC, to check purity (homo-PS formation) and composition homogeneity. Well-defined PS-b-PEO-b-PS triblock copolymers having a large range of chain dimensions (Mn of block copolymers ranging from 5000 to 200000 g/mol) and block lengths (Mn of PS blocks between 2000 and 83000 g/mol) with a low polydispersity index (1.1−1.3) were prepared using this new route.