In 2017 Cooktown resident Donna Steele was murdered and the case remained unsolved for more than a year. The forensic evidence from the investigation included two lengths of synthetic twine and a new protocol for the comparison of twine was developed to enhance the potential value of any evidence. The method was developed using 23 samples of similar twine collected across Australia. Traditional methods of physical and microscopic comparisons and polymer analysis by infrared spectroscopy were retained. Micro-spectrophotometry was used as an objective assessment of colour and was able to identify five groupings within the background samples. Measurements of hydrogen and carbon stable isotopic composition provided further delineation of the background samples. Combining traditional methods with micro-spectrophotometry and stable isotope measurements, the two case samples were found to be distinct from the background population and were indistinguishable when compared to each other.