BackgroundIn forensic investigation of alleged sexual crimes, presumptive semen detection test methods are commonly used to reach preliminary identification of seminal fluid in questioned samples. These methods are based on the detection of components of semen such as enzyme acid phosphatase (AP). Of these methods, the acid phosphatase identification method still remains the most reliable and widely used presumptive test due to high activity of the AP in seminal fluid. In standard AP test, Bretamine Fast Blue B (FBB) reagent is used. However FBB has been explicitly classified as carcinogenic. Although FBB has been handled safely over time, there is a need at the moment to develop a simple, readily available, reliable and efficient method for screening the presence of semen in any material collected as evidence in a sexual assault crime.Given the improved sensitivity of DNA profiling tests that have been introduced in to routine forensic casework over recent years, the need for improved sensitivity at this first stage of detection has never been higher. FindingsHere we highlight a simple method using readily available reagents in standard biochemical laboratory as a substitute for the standard AP test for seminal fluid identification from a crime scene. This method is based on the hydrolysis of sodium–p-nitrophenyl phosphate at pH 5.5 by the acid phosphatase to produce an intense yellow coloured complex in 15 seconds. ConclusionsThe method presented is sensitive, reliable, efficient and routinely used in standard biochemical and pathology laboratories for spectrophotometric analysis of alkaline phosphatase. It can be easily and readily applied as a preliminary test for identification of semen at a crime scene that involves sexual assault.