Assessment of gene expression is an essential and sensitive indicator of the biological activity and function of a certain gene. The expression profile of R2R3 MYB gene (Solyc02g067760) was investigated at ten development stages of tomato fruits (Lycopersicon esculentum) of Micro-Tom cultivar using three different approaches; quantitative RT-PCR, semi-quantitative RT-PCR and the promising Tomato Expression Atlas (TEA) bioinformatics tool. RNA was extracted from fruit tissues and used for synthesizing cDNA. Quantitative and semi-quantitative expression analyses of R2R3 MYB gene using specific primers consistently showed different expression patterns at the different development stages of tomato fruit. R2R3 MYB exhibited maximum expression at the anthesis stage, suggesting its potential involvement in the pollination and fruit set process. The gene expression was drastically reduced in all other studied stages after anthesis. In the same context, spatiotemporal expression of R2R3 MYB during the ten development stages of tomato fruit using TEA bioinformatics tool revealed a similar expression profile to that obtained by qRT-PCR and semi-qRT-PCR. Comparable results of the three approaches provide new insights into the assessment of tomato gene expression profiling in the presence of Actin gene (Solyc04g011500) as a normalizing expression reference. The obtained results confirm the potentially essential role of the R2R3 MYB in fruit set leading the future research into exploring techniques and stratify of overexpressing this gene in the relevant economic crops.