When juxtaposed with 2D cell culture models, multicellular tumor spheroids demonstrate a capacity to faithfully replicate certain features inherent to solid tumors. These include spatial architecture, physiological responses, the release of soluble mediators, patterns of gene expression, and mechanisms of drug resistance. The morphological and behavioural similarities between 3D-cultured cells and cells within tumor masses highlight the potential of these models in studying cancer biology and drug responses. The liquid overlay method, hanging drop technique, and ultra-low adhesion plates are among the various methods for generating tumor spheroids, each with its advantages and applications. Gene expression studies, employing advanced methods such as microarrays, suppression subtractive hybridization, qRT-PCR, and mass-spectrometry-based proteomics revealed distinct expression patterns in 3D spheroids compared to 2D cultures, uncovering upregulation and downregulation of genes associated with tumor development, metastasis, and drug resistance. Protein expression studies identified alterations in key signaling pathways, metabolic characteristics, and phosphorylation levels, highlighting the impact of 3D culture on cellular responses. This study explores genes and proteins expression variations in various cancer cell lines cultivated in 3D spheroids, shedding light on the complexity of interactions in a more tumor-mimicking environment. The fusion of these analytical approaches not only advances scientific understanding but also holds promise for the development of more effective cancer treatments.