Background: Background: Natural dyes are increasingly being used in biomedical applications because of their low toxicity, biodegradability, and sustainability compared to synthetic dyes. Traditional stains, such as eosin-nigrosin, used for sperm evalu- ation, raise concerns about chemical toxicity. This study evalu- ated the efficacy of a beetroot (Beta vulgaris)-derived natural dye as a non-toxic alternative dye for human sperm morphol- ogy assessment. Materials and Methods: Beetroot pigment was extracted by homogenizing fresh beetroot in 70% ethanol (1:5 w/v), followed by agitation for 24 h at 4°C. After centrifugation (3000 rpm, 15 min) and filtration (Whatman No.1), the extract was diluted to 20% (v/v) with normal saline for staining. Ten air-dried human sperm smears (from two donors; all cells were non-viable) were stained with beetroot solution for 15 min at room temperature, then rinsed with PBS (pH=7.4) without fixation. Sperm mor- phology was exclusively evaluated using the WHO criteria by brightfield microscopy (1000× magnification). Eosin-nigrosin- stained slides were used for comparison. Results: The beetroot dye successfully stained the sperm head, midpiece, and tail, providing distinct visualization and color contrast adequate for morphological differentiation according to the WHO guidelines. Importantly, the dye maintained struc- tural integrity without causing head swelling or significant arti- facts. Visual clarity for morphological assessment was compa- rable to that of traditional eosin-nigrosin staining. The staining method exhibited good stability. Conclusion: Beetroot extract has significant potential as a nat- ural, safe, and sustainable histological stain for human sperm morphology analysis. This offers an appropriate method for routine laboratory assessments. Further validation may support its integration into clinical practice.

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