Alternative splicing (AS) is a key regulatory process in eukaryotes. Despite its importance, the impact of AS on proteome diversity in plants remains largely unexplored. Traditional tryptic digestion has limitations in detecting junction peptides because evolutionarily conserved nucleotide usage at exon boundaries increases the frequency of lysine- and arginine-coding triplets at exon ends, making detection of exon–exon and exon–intron junctions challenging. To address this gap, we performed a comprehensive analysis of the Arabidopsis proteome using the protease AspN combined with extensive offline fractionation. AspN is an endoprotease that cleaves at the N-terminal side of aspartic acid residues. To further enhance detection of alternatively spliced proteins, we analyzed both wild-type plants and the AS mutant acinus pinin. This approach enabled identification of isoform-specific peptides—both annotated and previously unannotated—using in-house algorithms and rigorous filtering steps. Our findings demonstrate that alternative splicing contributes substantially to proteome diversity in plants.