For structure simplification and material conservation, three novel candidates of the gas-liquid-solid (GLS) separator were developed for the up-flow reactors, typically UASB and EGSB, used for wastewater treatment. These proposed GLS separation prototypes were designed on a completely different separation mechanism that the European prototype adopted. This mechanism was observed and analyzed both in laboratory experiments and in computational fluid dynamic (CFD)-based 3D simulation. The three new GLS separators could reduce more than 19% in the height of dimensions and 20% in material consumption compared with the classical GLS separator. Nineteen experimental runs with the laboratory-scale fabricated separators, including both purchased granular sludge and locally obtained floccular sludge. suggested that the GLS separation prototypes were as excellent in accomplishing the supposed separation task as the presently widely used traditional counterparts. For further in-depth and detailed internal flow fields and gas phase behavior investigation, 3D CFD simulations were employed. The simulation revealed clearly and accurately the flow fields and retention properties of solid particles and gas bubbles among the layers of the GLS separator’s baffles and was in good agreement with the laboratory experiment. Analysis of Variance (ANOVA) of the simulation results was conducted for stability evaluation and dynamic characteristics analysis, which may be useful in the GLS separator’s routine operation.