Background To make it efficient, health care waste (HCW) segregation has to be done at the source. It must be simple, safe, well explained and understood by the personnel. To maintain good practice, regular training systems for old and new staff members and continuous assessment are essential to assure a high quality of waste management. Our objective is to create a new practical tool to assess the practice of surgical team members at our university hospital regarding the segregation of HCW. Methods Surgical team members were divided into four groups and 9 subgroups. Nine different colors were assigned to each subgroup. Pictures of 30 surgical wastes were used instead of the items themselves. Each item was marked by one color from the population set of colors in a way that each subgroup of the sample has a complete set of the 13 wastes marked by the corresponding color. Five artificial empty waste containers were created using 5 colors. Sets were provided to the staff as they were entering the operating theater on the study days. Segregation of the items was done in an individual manner at the spot of the distribution. Data were collected into tables using Microsoft (MS) Excel 2016 and was checked twice. Descriptive statistic was used to analyze the data. Results 64 health care professionals participated and 832 items were segregated. The mean wrong segregation rate was 12.6%. The operating theatre technician, residents, visitors, anesthetists, non-specialized nurses, nurse anesthetists, and surgeons got respectively 92.3%, 85.6%, 84.6%, 84.6%, 81.7%, 79.8% and 78.1% correct segregations. Each visitor (medical students from other centers), operating theater technician, nurse anesthetist, non-specialized nurse, surgeon, anesthetist, and resident had a 7.7%, 7.7%, 2.5%, 2.3%, 1.7%, 1.9% and 0.6% risk of incorrect segregation. In total, each member had a 0.3% risk of wrong segregation. Conclusion To our knowledge, no similar study was done until now and no similar practice assessment method was used so far in the literature. We introduced a new experimental tool that permitted to conclude that the members of the team of our surgical theater have good knowledge reflected by the low rates of wrong segregations. The method was safe, fast, attracting and more comparable to the daily work rhythm at the department studied.