Context .- As our understanding of genomic alterations underlying solid tumor malignancies continues to evolve, molecular testing of tumor samples is increasingly used to guide therapeutic management. Next-generation sequencing (NGS) provides a novel platform for the simultaneous screening of multiple genes using small amounts of DNA. Several recent studies have described NGS mutational analysis using cytologic specimens. The cytopathologist’s role in specimen assessment and triaging is critical to effectively implementing NGS in routine cytology practice. Objectives .- To review the NGS experience and a variety of preanalytic factors that affect NGS success rates of cytologic specimens at our institution. Data Sources .- To evaluate cytology specimen adequacy rates for NGS, we reviewed a 14-month period of image-guided fine-needle aspiration and core needle biopsies, used for testing. In addition, we reviewed data from our previously published studies to evaluate preanalytic factors affecting NGS success in these specimens. Conclusions .- Identifying factors that affect NGS success rates in cytology specimens is crucial for a better understanding of specimen adequacy requirements and for proper use of limited-volume tissue samples. In our practice, which uses direct smears as well as cell block sections, NGS success rates in core needle biopsy and fine-needle aspiration samples are comparable. The chance of successful testing is further increased by procuring concurrent fine-needle aspiration and core needle biopsy samples. The type of glass slides used for direct smears and the method of tissue extraction affect our DNA yield. Validating a DNA input for cytology samples that is lower than that recommended by the manufacturer has significantly increased our NGS success rate.