Background: Despite its wide usage among healthcare workers (HCWs), Depression Anxiety Stress Scales 21 (DASS-21) has received scant attention in terms of its psychometric properties among HCWs. The present study examined the factor structure of DASS-21 among representative HCWs in Muscat Region, Oman. The related aim is to examine factorial invariance across samples of males and females. Methods: Confirmatory factor analyses were conducted to evaluate the latent structure of DASS-21. Factorial invariance between males and females was examined. The Cronbach alpha coefficients of DASS-21 were calculated to assess the internal consistency of the scale. Results: The CFA of the DASS-21 indicated that the three-factor solution best fits the data. The results also showed that DASS21 had overall good-to-excellent reliability. Multigroup analysis did not indicate gender differences. Limitations: The present research study was limited as the accrued sample was drawn from HCWs working in the urban area, the Muscat region. As the study was conducted during the COVID-19 pandemic, this has the potential to constitute potential confounder to hamper the generalization of the present study. Conclusions: The results supported the applicability of DASS-21 across cultures and sex. The present data provide initial support for the psychometric properties of Arabic DASS among HCWs.