Recently, the Ka/Ks ratio test, which assesses the protein-coding potentials of genomic regions based on their non-synonymous to synonymous divergence rates, has been proposed and successfully used in genome annotations of eukaryotes. We systematically performed the Ka/Ks ratio test on 925 transcript-confirmed alternatively spliced exons in the human genome, which we describe in this manuscript. We found that 22.3% of evolutionarily conserved alternatively spliced exons cannot pass the Ka/Ks ratio test, compared with 9.8% for constitutive exons. The false negative rate was the highest (85.7%) for exons with low frequencies of transcript inclusion. Analyses of alternatively spliced exons supported by full-length mRNA sequences yielded similar results, and nearly half of exons involved in ancestral alternative splicing events could not pass this test. Our analysis suggests a future direction to incorporate comparative genomics-based alternative splicing predictions with the Ka/Ks ratio test in higher eukaryotes with extensive RNA alternative splicing.