Background: Saliva is an important body fluid having importance in forensic tracing, is identified by its high amylase activity compared to other biological materials using salivary staining method. Whether this activity changes with oral habits, time and temperature, material on which the stain is absorbed or collected and in other biologic materials has to be known. Methodology: Study samples comprised of fresh saliva samples of 20 subjects each, with and without habits and one sample each of blood and vaginal secretions, collected on both filter paper and cotton cloth. Amylase activity of the pre-weighed blood and vaginal secretions, salivary samples at differing temperature and time and salivary samples diluted with blood & vaginal secretions at differing concentrations were recorded. Results: Saliva samples showed high specific activity of amylase in cloth and in habituated subjects, but this activity decreased with time and temperature. Whereas blood and vaginal samples showed very less specific activity compared to saliva samples and it decreased with increasing dilutions with saliva. Conclusion: When studied in a larger scale, the study observations may help in narrowing down the findings at a crime scene which could serve as a guide line for further research in forensics.