Background: Healthcare workers are at an increased risk of acquiring vaccine-preventable diseases and are known to be reliable sources of information for the patients and their relatives. Knowledge and attitudes of healthcare workers about vaccines are thus important determinants of their own vaccination uptake and their intention to recommend vaccinations to their patients. However, culturally adapted tools and studies to address vaccine uptake and hesitancy as well as related behaviors among healthcare workers in the Global South are limited. Methods: We propose a mixed methods project to understand the extent and determinants of vaccination hesitancy among healthcare workers and construct a validated scale to measure this complex and context-specific phenomenon in Cape Town. We will summarize responses as counts and percentages for categorical variables and means with standard deviations (or medians with inter quartile ranges) for continuous variables. We will run the Shapiro-Wilks test to assess the normality. Analysis of the variance, chi-square tests, and equivalents will be conducted as appropriate for group comparisons. Logistic regression models will also be performed to assess association between variables. We will focus on the seasonal influenza and COVID-19 vaccines. We will use an existing tool developed and validated in Germany and the United States of America to measure five psychological determinants of vaccination (referred to as the 5C scale), as the basis to develop and validate a scale to measure the scope and determinants of vaccine hesitancy and acceptance among healthcare workers in Cape Town. Discussion and conclusion: Through this study, we hope to expand the scientific evidence base on vaccination acceptance and demand among healthcare workers in South Africa and build resources to enable better understanding of, detection, and response to vaccination hesitancy in Cape Town.