Alternative splicing (AS) is an important regulatory process in eukaryotes and, in plants, is influenced by various stressors and developmental stages. Despite its significance, the impact of AS on proteome diversity in plants has not been fully explored. To address this gap, we investigated isoform-specific peptides in wild-type (WT) plants and acinus pinin double mutants. ACINUS and PININ are evolutionarily conserved splicing factors, and their disruption induces splicing events resembling those triggered by diverse abiotic and biotic stressors. We generated extensively fractionated samples from a pooled peptide mix containing five replicates each of WT and acinus pinin, labeled with TMT11. This dataset was integrated with additional data generated using the alternative protease AspN, as well as mined data from the Arabidopsis Proteome Draft (Mergner et al., 2020, Nature), to enhance isoform-specific peptide detection. Using in-house algorithms and tailored filtering steps, this approach enabled identification of isoform-specific peptides, both annotated and previously unannotated. Our findings demonstrate that alternative splicing contributes substantially to proteome diversity in plants.