Simple Summary During tumor evolution, heterogeneous structural and functional changes occur in the tumor microenvironment. These complex changes have pro- or anti-tumorigenesis effects and have an impact on therapy efficiency. Therefore, the tumor microenvironment needs to be non-invasively characterized over time. The aim of this preclinical work is to compare the sensitivity of modifications occurring during tumor evolution of volume, immunohistochemistry and non-invasive quantitative ultrasound parameters (Shear Wave Elastography and dynamic Contrast-Enhanced Ultrasound) and to study the link between them. The complementary evaluation over time of multiple morphological and functional parameters during tumor growth underlines the need to integrate histological, morphological, functional, and, ultimately, genomic information into models that can consider the temporal and spatial variability of features to better understand tumor evolution. Abstract Purpose: There is a clinical need to better non-invasively characterize the tumor microenvironment in order to reveal evidence of early tumor response to therapy and to better understand therapeutic response. The goals of this work are first to compare the sensitivity to modifications occurring during tumor growth for measurements of tumor volume, immunohistochemistry parameters, and emerging ultrasound parameters (Shear Wave Elastography (SWE) and dynamic Contrast-Enhanced Ultrasound (CEUS)), and secondly, to study the link between the different parameters. Methods: Five different groups of 9 to 10 BALB/c female mice with subcutaneous CT26 tumors were imaged using B-mode morphological imaging, SWE, and CEUS at different dates. Whole-slice immunohistological data stained for the nuclei, T lymphocytes, apoptosis, and vascular endothelium from these tumors were analyzed. Results: Tumor volume and three CEUS parameters (Time to Peak, Wash-In Rate, and Wash-Out Rate) significantly changed over time. The immunohistological parameters, CEUS parameters, and SWE parameters showed intracorrelation. Four immunohistological parameters (the number of T lymphocytes per mm2 and its standard deviation, the percentage area of apoptosis, and the colocalization of apoptosis and vascular endothelium) were correlated with the CEUS parameters (Time to Peak, Wash-In Rate, Wash-Out Rate, and Mean Transit Time). The SWE parameters were not correlated with the CEUS parameters nor with the immunohistological parameters. Conclusions: US imaging can provide additional information on tumoral changes. This could help to better explore the effect of therapies on tumor evolution, by studying the evolution of the parameters over time and by studying their correlations.