Current efforts to recover the Neandertal and mammoth genomes by 454 DNA sequencing demonstrate the sensitivity of this technology. However, routine 454 sequencing applications still require microgram quantities of initial material. This is due to a lack of effective methods for quantifying 454 sequencing libraries, necessitating expensive and labour-intensive procedures when sequencing ancient DNA and other poor DNA samples. Here we report a 454 sequencing library quantification method based on quantitative PCR that effectively eliminates these limitations. We estimated both the molecule numbers and the fragment size distributions in sequencing libraries derived from Neandertal DNA extracts, SAGE ditags and bonobo genomic DNA, obtaining optimal sequencing yields without performing any titration runs. Using this method, 454 sequencing can routinely be performed from as little as 50 pg of initial material without titration runs, thereby drastically reducing costs while increasing the scope of sample throughput and protocol development on the 454 platform. The method should also apply to Illumina/Solexa and ABI/SOLiD sequencing, and should therefore help to widen the accessibility of all three platforms.