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Background In order to reduce in vivo animal experiments in peripheral nerve regeneration research, in vitro models are desirable. Common two dimensional (2D) co-culture models lack the complex interactions of three dimensional (3D) physiological structures. The aim of the study was to establish a neuronal 3D spheroidal sprouting assay for peripheral nerve regeneration. New method Spheroids consisting of Schwann cells (SC, 500 cells/spheroid) and NG108-15 cells (NG, 50 cells/spheroid), a hybrid cell line, were formed in hanging drops and were embedded in a 3D collagen matrix. Spheroid sprout lengths were compared to those of the neurites of NG in a 2D co-culture with SC. Lengths were measured using phase contrast images taken every day over 10 days. Additionally we took fluorescence images to visualize the PKH26-labeled NG in both culture systems. Results Initially thin neurites grew out in both co-cultures, over time the sprouts’ diameter in the 3D culture increased. The direct comparison of the sprout length revealed significantly longer neurites in the 3D co-culture from day 7 until day 10 ( p < 0.001). Comparison with existing methods Other co-culture models either display processes in 2D or need complex matrices to create 3D structures. Our spheroidal model is easy to establish, highly flexible and nevertheless 3D. Conclusions The 3D-Schwann cell-neuron spheroid model shows that by simply transferring a 2D into a 3D co-culture with multiplication of cell-cell contacts, a significant increase of neurite length can be achieved. The model is a relatively simple method for the investigation of neurite development in vitro. [ABSTRACT FROM AUTHOR]