Background: Defilement has been recognized as one of the crimes in which such a ceiling in evidential presentation is critical. According to WHO statistics, More than 95 million children are abused annually, Where Africa is the most affected continent. Defilement incidences are alarming globally, regionally, and locally. There are new cases of defilement reported every week with 83 % emanating from a well-known perpetrator to the survivor. Objective: The study aimed to assess the use of medical forensic evidence in the investigation and prosecution process of defilement perpetrators, A case study of Ruiru Sub-County, Kenya. Method: Research was conducted using an analytical cross-sectional approach. Sixty-six participants in the study were chosen using a stratified random sampling technique. The techniques used to collect the data were both qualitative and quantitative. The strength of the relationship between the dependent and independent variables was assessed using the Pearson correlation. The threshold for statistical significance was set at 0.05. Results: There was a significant correlation between Compliant with set standards against investigation and the prosecution of defilement incidences(p=.005). There was a significant Correlation between the Availability of forensic tools against investigation and the prosecution of defilement incidents(p=.000). Concerning the correlation between knowledge of the guardians against investigation and prosecution of defilement Incidents there was no significant association(p=.630). Conclusion: From this research, compliance with set standards and procedures for evidence admissibility of defilement evidence was adequate. Study respondents had good knowledge of the use of medical forensic evidence during the investigation and prosecution of defilement incidences. The study concluded that most of the facilities and officers are not equipped with materials for the collection of evidence.