Although lab-on-a-chip (LoC) enables the performance of experiments on a small scale using miniaturized devices, ability to detect dynamic cellular behaviour over long periods of time (e.g days or weeks) within 3D microenvironment is still in its infancy. The integration of optical detection techniques in microfluidic devices are often robust and sensitive for the assessment of cell cultures that are cultured in 3D environment. We have investigated, light scattering as a robust techniques capable of non-invasively and continuously determining cell morphology, proliferation, viability and migration of 3D cell cultures. Although light scattering has successfully been employed to study a variety of cellular responses of two-dimensional microfluidic cell cultures systems over recent years, its applications for 3D cell cultures systems has not been investigated in detail. 3D cell cultures system has the benefit that it mimics a suitable microenvironment for the growth of cells which usually reside in tissue surrounded by extracellular matrix. Their maturation into specialized cell types occurs in a more natural manner morphologically and physiologically compared to cells that are cultured in 2D cultures. In an attempt to show the potential benefit of laser scattering (LS) system for hydrogel-based microfluidic 3D cell culture analysis, the presented work investigates strengths, limitations and risk factors associated with the interpretation of data. Consequently, recently established, the LS system with the source wavelength of 488nm is characterized in detail by utilizing polystyrene particles of various sizes in PBS and in 3D-hydrogels. Additionally, LS is evaluated for cell culture analysis using mouse embryonic fibroblasts and Jurkat cells which are embedded in hydrogels. Results of the study revealed the feasibility of the LS system as reliable detection method for organ-on-a-chip applications. Practical application using chemotactic lymphocyte migration study revealed that LS is ideally suited to non-invasively and label free probe 3D-hydrogel based cell culture systems.