Sanger DNA sequencing is a robust and flexible technology and should have a crucial role in clinical practice for a long time. Nevertheless, in routine application of DNA sequencing, we are regularly confronted with sequence data quality problems. Surprisingly, we found that the definition of sequence quality is fuzzy and too empirical for many clinical applications. There are few studies or guidelines that directly address quality issues for Sanger sequencing in clinical situations. In addition, these use several combined parameters to ensure the sequence quality; this is too complicated to apply for daily use. Our heuristic analysis of nearly 46,000 sequence traces demonstrated that a combination of three basic parameters (average quality value, average sequence intensity, and electropherogram profile) was necessary and sufficient to determine accurately the quality of any sequence, even when deletions, insertions, and/or repeat sequence regions were present in a sequence trace. Therefore, we propose a simple and practical method with a diagram and decision making table for sequence quality determination in clinical sequencing.