Transfected cell microarray is a promising method for accelerating the functional exploration of the genome, giving information about protein function in the living cell. The microarrays consist of clusters of cells (spots) overexpressing or silencing a particular gene product. The subsequent analysis of the phenotypic consequences of such perturbations can then be detected using cell-based assays. The focus in the present study was to establish an experimental design and a robust analysis approach for fluorescence intensity data, and to address the use of replicates for studying regulation of gene expression with varying complexity and effect size. Our analysis pipeline includes measurement of fluorescence intensities, normalization strategies using negative control spots and internal control plasmids, and linear regression (ANOVA) modelling for estimating biological effects and calculating P-values for comparisons of interests. Our results show the potential of transfected cell microarrays in studying complex regulation of gene expression by enabling measurement of biological responses in cells with overexpression and downregulation of specific gene products, combined with the possibility of assaying the effects of external stimuli. Simulation experiments show that transfected cell microarrays can be used to reliably detect even quantitatively minor biological effects by including several technical and experimental replicates.