Gradient hydrogels are functionally graded biomaterials that over the last decades have garnered significant attention as valuable materials for tissue repair and regeneration. This study elucidates key factors crucially determining the character or pattern of a buoyancy-driven gradient in agarose hydrogels and also provides fundamental information regarding the complex interpretation of the formed gradient hydrogels obtained by the different characterization techniques, including rheology, UV-Vis spectroscopy, FCS, AFM, SEM. The employed techniques were successfully optimized to enhance the understanding of the gradient properties of these hydrogels. Demonstrated results showed that the agarose concentration gradient could be successfully controlled and tailored, which offers a valuable contribution to the design of gradient hydrogel. Moreover, a novel method for determining the average agarose concentration along the gradient axis is presented, improving the precision of gradient characterization. The integration of these approaches significantly enhances the comprehension of the internal structure and properties of gradient materials, contributing to advancements in their understanding and application.