Introduction: The most effective way to break the chain of transmission of infection in healthcare facility is Hand Hygiene (HH). However, the HH adherence rate/compliance is varying worldwide. Healthcare facilities should follow comprehensive, systematic approach for assessment of HH adherence rate. Periodic monitoring and necessary feedback is critical to improve HH adherence rate/compliance. Aim: To know the impact of the HH training of Healthcare Workers (HCW) on HH adherence rate. Materials and Methods: This prospective study was conducted at SMS Multispecialty Hospital, Ahmedabad, Gujarat, India. The study was conducted for the duration of six months (October 2019-March 2020). Total 416 opportunities were accessed in two sessions {203 pretraining and 213 post-training} among different HCWs of ICU and NICU. HH adherence rate was measured by direct observation methods by trained, non participating neutral person as per World Health Organisation (WHO) HH guidelines. HH training was given to HCWs (doctors, nursing staff and attendant) of Intensive Care Unit (ICU) and Neonatal Intensive Care Unit (NICU). Pre and post-training HH adherence rate was calculated among HCWs of ICU and NICU in respect to WHO’s five movement of HH. Chi-square test was used to do statistical analysis. Results: Pre and post-training HH adherence rate of HCWs of ICU is 41% and 69% respectively. However, pre and post- training HH adherence rate of HCWs of NICU was 52% and 75%, respectively. Both ICU and NICU post-training HH adherence rate of HCWs was improved which was statistically significant (p-value <0.00001). Both pre and post-training, compared to doctors, nursing staff had better HH adherence rate (45%, 67% in ICU and 58% and 78% in NICU). Hand rub was preferred method of HH (93%). Conclusion: The HH training improved the HH adherence rate of HCWs of ICU and NICU. Induction and periodic training of HCWs improves HH adherence rate of HCWs.