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Abstract Background and aim Fear of coronavirus disease 2019 (COVID-19) and its complications may result in stigmatization of individuals who may carry the virus. This is of special concern to healthcare workers who tolerate additional physical and emotional stress at times of pandemic. The aims of this study are to (1) develop and validate the COVID-19 Stigma Scale (COVISS-HCWs) survey; and (2) investigate the experience of stigma among healthcare workers possibly dealing with COVID-19 patients in five major public hospitals in Damascus, Syria. Methods We divided the sample into two parts and then underwent EFA on the first 350 participants, dividing the 14 questions into two dimensions. Furthermore, CFA was conducted on the other 350 participants to confirm how correctly a hypothesized model matched the factor structure by EFA, as described above. Moreover, the coefficient of determination (R2) and item–scale correlations (standardized factor loading) were estimated to establish the acceptability of the final structure of the COVISS-HCWs. Through a cross-sectional study, a convenience sample of 700 healthcare workers participated in a self-administered questionnaire containing a section for demographic variables and another for newly designed COVISS-HCWs. The scale comprises 14 adapted and novel items that measure two subscales: feelings of perceived harm and inferiority, and avoidance. Descriptive statistics, reliability, and validity were evaluated. Results The 14 COVISS-HCWs items were reduced to 11 items with a high Cronbach’s α of 0.909. A significant correlation was observed between the responses to each COVISS-HCWs item and the corresponding subscale, and between each subscale and the overall scale. Feeling stigmatized was reported by 9.86% of the participants. Younger age, low socioeconomic status, and higher intensity of contact with COVID-19 patients significantly correlated with higher stigmatization. Conclusions The novel COVISS-HCWs is a reliable and valid tool to evaluate stigma among healthcare workers during the COVID-19 pandemic. The Stigma prevalence among healthcare workers was 9.86%. Therefore, this must be addressed to prevent possible psychosocial and public health repercussions.