The known regulatory role of 3' untranslated regions (3'UTRs) and poly(A) tails in RNA localization, stability, and translation, and polyadenylation regulation defects leading to human diseases such as oculopharyngeal muscular dystrophy, thalassemias, thrombophilia, and IPEX syndrome underline the need to fully characterize genome-wide polyadenylation states and mechanisms across normal physiological and disease states. This chapter outlines the quantitative polyadenylation site mapping and analysis strategies developed with the single-molecule direct RNA sequencing technology.