A fluorescence microscopy considered as a powerful imaging tool in biology and medicine. In addition to useful signal obtained from fluorescence microscopy, there are some defects in its images such as random variation in brightness, noise that caused by photon detection and some background pixels in the acquired fluorescence microscopic images appear wrongly auto-fluorescence property. All these practical limitations have a negative impact on the correct vision and analysis of the fluorescent microscope users. Our research enters the field of automation of image processing and image analysis using image processing techniques and applying this processing and analysis on one of the very important experiments in biology science. This research is devoted to develop a system based on digital image processing methodology to localize and assess the concentration of saponins accumulation in plant tissues using Fluorescence microscopic image. The proposed system involved preprocessing steps than to make the region of interest more obvious and reflects the saponins accumulation area. Also, the system introduces a simple mathematical way for concentration assessment, and it was justified through the test results. It includes building a system to get microscopic images with best appearance and no defects. It determines the saponins accumulation sites in leaves, rhizomes and shoot apex of Y. gloriosa Variegata and their in vitro cultured tissues (Calli, direct and indirect regenerated shoots and rhizomes/roots). Statistical analysis is performed using a computer to get the mean and median of saponins intensities in each part, and finally perform a comparison between them to determine which part can record the highest intensity level of saponins. The results showed high ability of the system to determine the locations and intensity of saponins in the different parts of the plant. It performs the statistical analysis very quickly. In in vitro culture, it was found that callus treated with Thidiazuron (TDZ) in a combination with benzyl aminopurine (BA) and naphthyl acetic acid (NAA) after 3 weeks of culture had the highest level of saponins accumulation, while the leaves of intact plant recorded the second highest accumulation of saponins.