Mass spectrometry (MS) has emerged as an indispensable tool in plant biology, offering exceptional versatility for investigating protein-level responses to environmental stimuli. This chapter provides an overview of quantitative proteomics applied to plant breeding, covering the experimental workflow, sample preparation, and data analysis. Special attention is given to experimental design, emphasizing the importance of an adequate number of biological replicates and choosing the appropriate proteomics approach according to the comparisons to be analyzed. Although pairwise comparisons are common, time-resolved and dose-dependent designs are increasingly preferred for their robustness in revealing co-accumulated proteins and reducing uncertainty in determining biomarkers. This chapter highlights the power of proteomics to elucidate plant responses to abiotic and biotic stresses, and heterosis, demonstrating its ability to uncover signaling pathways, regulatory proteins, and stress- and trait-specific biomarkers. An outlook on future directions is provided, highlighting the transformative potential of proteomics in plant science and crop improvement.