Microindentation of fresh biological tissues is necessary for the creation of 3D biomimetic models that accurately represent the native extracellular matrix microenvironment. However, tissue must first be precisely sectioned into slices. Challenges exist in the preparation of fresh tissue slices, as they can tear easily and must be processed rapidly in order to mitigate tissue degradation. In this study, we propose an optimised mounting condition for microindentation and demonstrate that embedding tissue in a mixture of 2.5% agarose and 1.5% gelatin is the most favourable method of tissue slice mounting for microindentation. This protocol allows for rapid processing of fresh biological tissue and is applicable to a variety of tissue types.