Advancements in DNA sequencing technologies with decreasing costs have sparked the generation of larger gene expression datasets generated at an accelerating rate. The study aimed to visualize the spatiotemporal profiles of the tomato (Solanum lycopersicum L.) genes involved in L-proline biosynthesis and to show their potential functions. Increasing L-proline accumulation, through upregulation and downregulation of genes responsible for L-proline biosynthesis and degradation, plays an essential role in tomato plants suffering abiotic and biotic stress. Understanding the possible mechanism of L-proline biosynthesis and degradation needs an urgent study of the expression pattern and function of genes involved in these physiological processes. The study identified the genes governing the L-proline biosynthesis and degradation pathways and their expression profiles in 10 stages of tomato fruit development using the Tomato Expression Atlas (TEA) bioinformatic tool. The analysis showed that L-proline biosynthesis resulted from three pathways governed by six genes, while its degradation occurred in two pathways managed by three genes. The bioinformatics analysis also showed the expression of proline synthesis/degradation-related genes in fruit parts at various developmental stages. However, proline degradation-related genes showed higher expression levels than biosynthesis-related genes. This study sheds light on a recent bioinformatics tool, which will pave the way to detect early plant performance by analyzing the expression profiles of genes.