AFM-based rheology methods enable the investigation of the viscoelastic properties ofcancer cells. Such properties are known to be essential for cell functions, especially for malignantcells. Here, the relevance of the force modulation method was investigated to characterize theviscoelasticity of bladder cancer cells of various invasiveness on soft substrates, revealing that therheology parameters are a signature of malignancy. Furthermore, the collagen microenvironmentaffects the viscoelastic moduli of cancer cell spheroids; thus, collagen serves as a powerful proxy,leading to an increase of the dynamic moduli vs. frequency, as predicted by a double power lawmodel. Taken together, these results shed new light on how cancer cells and tissues adapt theirviscoelastic properties depending on their malignancy and the microenvironment. This method couldbe an attractive way to control their properties in the future, based on the similarity of spheroidswith in vivo tumor models.