The availability of high-quality germline DNA is an important prerequisite for a variety of genetic analyses. We have shown that fingernail clippings provide an optimal source of autologous, constitutional DNA for PCR-based applications. However, most existing protocols for nucleic acid purification from nails do not provide sufficiently high yields of pure and intact DNA for more demanding downstream analyses such as next-generation sequencing. We extensively tested and systematically modified a number of different protocols for DNA purification from nail material to optimize the yield and quality. The integrity of DNA was determined by PCR amplification of short (<300 bp), mid-range (>400 bp), and long-range (>2 kb) sequences by using different target genes. Among the methods tested, the Prepfiler Forensic DNA extraction kit was identified as the most appropriate approach to isolate high-quality DNA from nail clippings. A standardized input of 20 mg of nail material (1 to 10 pieces of fingernail clippings) yielded a mean of 1 μg of DNA (range, 0.5 to 2.3 μg). Subsequent PCR analysis indicated efficient amplifiability of short- and mid-range targets in 93% and 90% and long-range fragments in 60% of the samples tested. The adequacy for next-generation sequencing applications was found by successful high-resolution histocompatibility leukocyte antigen typing in 10 patients who received a transplant. Hence, the protocol presented facilitates the exploitation of fingernail material even for demanding genomic analyses both in research and diagnostics.