Aqueous polymer solutions exhibiting a lower critical solution temperature (LCST) in the physiological range are widely used in biomedical applications. Of particular interest are polymers that contain additional reactive groups for further conjugation of drugs, dyes, or enzymes. For specific applications, detailed knowledge and understanding of the phase transition behavior (e.g., phase separation, transition range, and dehydration on the micro- and macroscopic level) and its dependence on various intrinsic (molecular weight and polymer functionalization) and extrinsic (polymer concentration and salt presence) factors are critical. In this context, we present a comprehensive study of the thermoresponsive properties of two unprecedented glycerol ether-based poly(meth)acrylates with β-hydroxy-functional side chains, namely, the structurally isomeric poly(3-ethoxy-2-hydroxypropyl)acrylate (pEHPA) and poly(2-hydroxy-3-methoxypropyl methacrylate) (pHMPMA). The distinct amphiphilic balance of pEHPA with a higher side chain hydrophobicity resulted in lower cloud point temperatures (22–33 °C), while shifting hydrophobicity to the backbone in pHMPMA led to increased cloud point temperatures (37–67 °C), accompanied by higher sensitivity of the phase transition to intrinsic and extrinsic factors. Turbidimetry, dynamic light scattering, and NMR measurements revealed that the hydration of β-hydroxy side chains primarily governs the transition behavior, resulting in distinct phase separation mechanisms between the two polymer types. Based on this knowledge, the rational design of hydroxy groups presenting poly(meth)acrylates with adjustable hydration becomes feasible. Cyanine5 (Cy5)-labeling of the hydroxy groups and temperature-dependent fluorescence analysis demonstrated the potential of these polymers as postfunctionalizable thermoresponsive polymer platforms, e.g., for bioseparation.