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Background: Hydroxychloroquine had attracted significant attention in the initial phases of the COVID-19 pandemic but current recommendations do not support its use. However, the evidence against its use as pre-exposure prophylaxis have been of low to moderate quality and have been limited by high risk of bias., Methods: Following institutional ethics committee approval, healthcare workers (n = 1294) completing their first week-long COVID in-patient duty, subsequent institutional quarantine and RT-PCR testing for COVID-19 infection were included for this prospective cohort study. Demographic data, hydroxychloroquine usage and related adverse effects were captured through a 'Caring for the Caregivers' surveillance system. A chi-Square test of independence was used to determine the effect of hydroxychloroquine prophylaxis., Results: Among the 1294 participants (age: 31 ± 7 years, 61% women), 273 (21.1%) healthcare workers used hydroxychloroquine prophylaxis as per Indian Council of Medical Research recommendations and 83/1294 (6.4%) tested positive after their duty. There was no significant difference in COVID-19 incidence between those on hydroxychloroquine prophylaxis and those not on it (5.9% vs 6.6%, χ 2  = 0.177, p = 0.675; RR = 0.89, 95% CI - 0.53 to 1.52). There were no significant adverse effects to hydroxychloroquine usage., Conclusion: This study demonstrated no benefit of hydroxychloroquine prophylaxis and provides quality evidence against its use in COVID-19 prevention.