Microarray image processing leads to the characterization of gene expression levels simultaneously, for all cellular transcripts (mRNAs) in a single experiment. The calculation of expression levels for each microarray spot/gene is a crucial step to extract valuable information. By measuring the mRNA levels for the whole genome, the microarray experiments are capable to study functionality, pathological phenotype, and response of cells to a pharmaceutical treatment. The processing of the extensive number of non-homogeneous data contained in microarray images is still a challenge. We propose a density based spatial clustering procedure driven by a level-set approach for microarray spot segmentation together with a complete set of quality measures used to evaluate the proposed method compared with existing approaches for gene expression levels estimation. The set of quality measures used for evaluation include: regression ratios, intensity ratios, mean absolute error, coefficient of variation and fold change factor. We applied the proposed image processing pipeline to a set of microarray images and compared our results with the ones delivered by Genepix, using the aforementioned quality measures. The advantage of our proposed method is highlighted by a selection of up-regulated genes that had been identified exclusively by our approach. These genes prove to add valuable information regarding the biological mechanism activated as a response of Arabidopsis T to pathogen infection.