Despite intensive research efforts, which have significantly improved melanoma patient survival, therapy resistance to the targeted mono- and combined therapies remains unsolved problems. The major focus of our study was to investigate molecular alterations associated with acquired resistance in malignant melanoma cell line models. It has been observed that the gene expression pattern of cells is altered during conventional cell culturing due to the unlimited access to oxygen, metabolites, nutrients, and signalling molecules. One aims of our study was to generate reproducible three-dimensional melanoma spheroid models from BRAF inhibitor sensitive and resistant melanoma cell lines and compare the gene expression signatures of the differently cultured melanoma cells. Using Affymetrix Human Gene arrays, we determined the gene expression pattern of melanoma cell lines. We found a large number of differentially expressed genes between drug-sensitive cells grown under different cell cultures. Pathway analysis showed that the differently expressed genes were mainly associated with cell cycle, p53, and other cancer-related pathways. Drug-resistant cells grown under 2D and 3D cell culture conditions exhibited 297 differentially expressed genes (72 up- and 225 downregulated) and were linked to various pathways including cellular translation, axon guidance, and IGF1R signalling. In addition, 13 genes, including DCUN1D1, CMSS1, ZNF639, ABHD4, and HIST1H2BB, exhibited inverse expression between sensitive- and resistant spheroid, with functional role in anoikis resistance and cell cycle regulation. In parallel, we explored molecular alterations associated with acquired resistance during combinatorial treatment of BRAFV600E mutant melanoma cell lines using BRAF and MEK inhibitors. After establishing six melanoma cell lines that became resistant during encorafenib (BRAFi) and binimetinib (MEKi) treatment we evaluated the invasive properties, studied the effect of “drug holiday” on cell proliferation and protein expression in the drug-sensitive and resistant cell lines, investigated the gene expression characteristics underlying BRAFi/MEKi resistance using RNAseq analyses and defined the biological functions of the differently expressed genes associated with the development of acquired resistance. We found that resistant cells changed their phenotype and had a higher invasive potential than sensitive cells. We observed that resistant cells did not develop drug dependency, which is in agreement with a recently published clinical study that suggests intermittent dosing might not be as beneficial as regular treatment. Using the Proteome Profiler Oncology Array, we identified cancer-related proteins that were differently expressed across sensitive and resistant cell lines, with no similar pattern among the cell lines. Furthermore, transcriptome analysis revealed that the 1591 differentially expressed genes (1024 up- and 567 down-regulated genes) are functionally linked to a variety of biological functions, including epithelial-mesenchymal transition, and KRAS signalling, that may lead to resistance. In summary, our data provide the first insight into differently expressed genes that might be involved in spheroid formation in BRAFi sensitive and resistant melanoma cells. The current data on the development of acquired resistance using combination of BRAF and MEK inhibitors, offer the first understanding into differentially expressed genes and provide protein expression patterns associated with a BRAFi/MEKi-resistant phenotype in melanoma cells. Our findings contribute to a better understanding of the complex mechanisms leading to acquired resistance during combined treatment of BRAF-mutant melanoma. However, further studies are needed to identify the key molecules and signalling pathways responsible for therapeutic escape during BRAFi/MEKi treatment and to prevent the initiation of acquired drug resistance in melanoma.