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Abstract Objective: To describe utilization of at-home coronavirus disease 2019 (COVID-19) testing among healthcare workers (HCW). Design: Serial cross-sectional study. Setting and participants: HCWs in the Chicago area. Methods: Serial surveys were conducted from the Northwestern Medicine (NM HCW SARS-CoV-2) Serology Cohort Study. In April 2022, participants reflected on the past 30 days to complete an online survey regarding COVID-19 home testing. Surveys were repeated in June and November 2022. The percentage of completed home tests and ever-positive tests were reported. Multivariable Poisson regression was used to calculate prevalence rate ratios (PRR) and univariate analysis was used for association between participant characteristics with home testing and positivity. Results: Overall, 2,226 (62.4%) of 3,569 responded to the survey in April. Home testing was reported by 26.6% of respondents and 5.9% reported having at least one positive home test. Testing was highest among those 30–39 years old (35.9%) and nurses (28.3%). A positive test was associated (P < .001) with exposure to people, other than patients with known or suspected COVID-19. Home testing increased in June to 36.4% (positivity 19.9%) and decreased to 25% (positivity 13.5%) by November. Conclusion: Our cohort findings show the overall increase in both home testing and ever positivity from April to November – a period where changes in variants of concern of SARS-CoV-2 were reported nationwide. Having an exposure to people, other than patients with known or suspected COVID-19 was significantly associated with both, higher home testing frequency and ever-test positivity.