Objective: Buccal epithelial cells in saliva traces found at a crime scene can be used for sex determination by examining the presence of Barr bodies in the nucleus. Papanicolaou (PAP) and fluorescent stains, among others, have been used in the past. Aceto-orcein (AO) is a relatively unexplored staining technique for this purpose. This study aims to assess the efficacy of sex determination using AO and PAP stains for the detection of Barr bodies in buccal mucosal scrapes. Study Design: Buccal scrapings were collected from 120 healthy individuals (60 males and 60 females). They were stained with AO and PAP. Fifty cells in each sample were analyzed for identification of Barr bodies. Samples with a presence of Barr bodies ≤5% were recorded as male and those with >5% were recorded as female. The percentage accuracy in determining sex using both stains was evaluated. Results: The percentage of Barr bodies in AO-stained slides ranged from 5 to 32 among females and from 0 to 8 in males, while with PAP the ranges recorded were 4-20 in females and 0-5 in males (p < 0.001). The sensitivity and specificity of AO for detecting sex accurately was around 98.3 and 95% for PAP. Conclusion: Sex determination using Barr bodies in the buccal cells is a simple method that provides up to 95-98% accuracy; making it a significant accessory for sex determination. AO proved to be a better stain than PAP for visualizing nuclear details, and its staining time was remarkably shorter. It also demonstrated enhanced sex estimation efficacy compared to PAP.