3′ end formation of eukaryotic messenger RNAs (mRNAs) is an essential process that influences mRNA stability, turnover, and translation. Polyadenylation is the process by which mRNAs are cleaved at specific sites in response to specific RNA sequence elements and binding of trans-acting protein factors; these cleaved mRNAs subsequently acquire non-templated poly(A) tails at their 3′ ends. Alternative polyadenylation occurs when multiple poly(A) signals are present in the primary mRNA transcript, in either the 3′ untranslated region (3′UTR) or other sites within the mRNA, resulting in multiple transcript variants of different lengths. We demonstrate here a new method, termed RHAPA (RNase H alternative polyadenylation assay), that employs conventional RT–PCR with gene-specific oligonucleotide hybridization and RNase H cleavage to directly measure and quantify alternatively polyadenylated transcripts. This method gives an absolute quantified expression level of each transcript variant and provides a way to examine poly(A) signal selection in different cell types and under different conditions. Ultimately, it can be used to further examine posttranscriptional regulation of gene expression.