Hydrogen sulphide (H 2 S) has emerged as an important signalling molecules due to its unique physiological and pathophysiological role in biological systems. H 2 S can be used as a biomarker for various diseases such as inflammation, cancer, cardiovascular, neurological and gastrointestinal disease. In this work, we report H 2 S chip which is designed for separation of all pools of H 2 S from plasma samples. The chip has been designed, fabricated and characterized. It operates with liberation, separation and trapping of H 2 S from plasma introduced to the chip.It consists of three distinct layers of polydimethylsiloxane (PDMS) structures which were bonded using 3M™ Transfer Tape. The releasing layer provides reaction surface for the liberation of H 2 S from the plasma sample. Releasing buffers allow release of various pools of H 2 S, including free, acid-labile and total sulphide. A silicone membrane is sandwiched between releasing and trapping layer. It worked as a H 2 S selective membrane where gas diffusion takes place from the releasing layer to the trapping layer. The trapping layer is H 2 S collection chamber. The full-chip performance was successfully characterized by derivatization of sulphide with excess MBB and analysing fluorescent product, sulphide dibimane (SDB) using the reverse-phase high-performance liquid chromatography (RP-HPLC) and fluorescence detection with eclipse XDB-C18 column. The sulphide transfer data showed 40% free sulphide, 38% acid-labile and 10% total sulphide transfer happens at 15 min. Performance of the chip was attributed to buffer, chip bonding technique and a silicon membrane that allows selective diffusion of hydrogen sulphide.