Parkinson’s Disease (PD), which exhibits a rapidly developing pathology, is one of the most devastating neurodegenerative diseases. To understand its molecular and cellular mechanisms and to attain a truly effective treatment, it is essential to develop standard, rapid, and reliable in vitro testing platforms for diseases. Classical two-dimensional (2D) cell culture is the starting point for the study of PD diagnosis and treatment. However, 2D grown cells do not exhibit the physiological properties of native tissues and provide limited data for testing drugs in vitro and understanding the mechanisms of diseases. Therefore, realistic 3D models similar to human physiology are required. In this study, the 2D and 3D PD modeling potentials of two cell lines (SHSY5Y and PC12), which are frequently used in neural tissue engineering studies, were compared. PD models generated by SHSY5Y and PC12 cells were evaluated by lactate dehydrogenase (LDH), Live&Dead, immunofluorescence, and quantitative reverse transcription polymerase chain reaction (qRT-PCR) analyses. It was determined that PC12 cells had weaker adherence properties than SHSY5Y cells, and therefore, SHSY5Y cells had higher microtissue formation potential. PC12 cells completely lost their dopaminergic properties in 3D conditions, whereas 6-OHDA applied SHSY5Y microtissues showed PD markers related to neurotoxicity. A practical and useful 3D disease model reflecting the characteristics of PD with SHSY5Y cells is presented.