5-Bromo-2-deoxyuridine (BrdU) staining is often used to evaluate cortical layer formation during mammalian brain development. This method allows the quantification of newly generated cells and therefore the study of the effects of xenobiotics or genetic factors on proliferation, cell death and migration behavior in a quantitative manner. However, these endpoints are generally assessed by time-consuming manual evaluation. In the present work, we introduce a novel procedure to identify and quantify BrdU+ cells within cortical layers, using the commercially available vHCS-Scan V.6.3.1 software to identify BrdU+ cell coordinates and the novel program ‘BrdeLuxe’ to define cortical layers and quantitatively assign BrdU+ cells to them. This procedure is compared to BrdU+ cell counting with the freeware ‘ImageJ’ in respect to the manual evaluation, all by two different researchers. BrdeLuxe shows high accuracy and precision for the determination of total number of BrdU+ cells compared to the manual counting, while ImageJ does not reach such results. Accuracy and precision are also higher for employing the BrdeLuxe program to evaluate the percentage of BrdU+ cells per brain layer compared to ImageJ. In terms of running time, BrdeLuxe is the fastest method of the three making it more suitable for multiple brain slices analyses.