Casein is a natural milk protein that has been investigated in drug delivery, cartilage tissue engineering, and other biotechnological applications. In particular, casein nanofibers are of high interest for tissue engineering. Electrospinning casein is usually performed by co-electrospinning it with a spinning agent, such as poly(vinyl alcohol), poly(caprolactone) (PCL), or poly(ethylene oxide) (PEO). Spinning casein solely has been found to be challenging due to its unsuitable viscoelasticity and extensive intermolecular interactions. Our study aims to optimize casein/PEO spinning solutions for needleless electrospinning in the wire-based electrospinning device Nanospider Lab. For this, PEO with different molecular weights (6 kDa-1 MDa) was mixed with different caseins in different ratios to prepare an aqueous spinning solution whose pH value was varied in the test series of experiments. Electrospinning duration (30 min) and nozzle size (0.9 mm) were kept constant, while the other spinning parameters, as well as the relative humidity in the spinning chamber, were modified. Our study reveals the impact of these parameters on the spinnability of the casein/PEO spinning solutions and the macroscopic homogeneity of the resulting nanofiber mats. Additionally, scanning electron microscopy (SEM) images show the impact of these parameters on the diameter distribution and morphology of the nanofibers. This poster gives the first overview of the optimum spinning parameters for the wire-based electrospinning of casein/PEO nanofiber mats.