Sulfane sulfur has been recognized as a common cellular component, participating in regulating enzyme activities and signaling pathways. However, the quantification of total sulfane sulfur in biological samples is still a challenge. Here, we developed a method to address the need. All tested sulfane sulfur reacted with sulfite and quantitatively converted to thiosulfate when heated at 95 °C in a solution of pH 9.5 for 10 min. The assay condition was also sufficient to convert total sulfane sulfur in biological samples to thiosulfate for further derivatization and quantification. We applied the method to detect sulfane sulfur contents at different growth phases of bacteria, yeast, mammalian cells, and zebrafish. Total sulfane sulfur contents in all of them increased in the early stage, kept at a steady state for a period, and declined sharply in the late stage of the growth. Sulfane sulfur contents varied in different species. For Escherichia coli, growth media also affected the sulfane sulfur contents. Total sulfane sulfur contents from different organs of mouse and shrimp were also detected, varying from 1 to 10 nmol/(mg of protein). Thus, the new method is suitable for the quantification of total sulfane sulfur in biological samples.