Background The workload in health care is increasing and hence, mental health issues are on the rise among health care professionals (HCPs). The digitization of patient care could be related to the increase in stress levels. It remains unclear whether the health information system or systems and digital health technologies (DHTs) being used in health care relieve the professionals or whether they represent a further burden. The mental construct that best describes this burden of technologies is mental workload (MWL). The measurement methods of MWL are particularly relevant in this sensitive setting. Objective This review aimed to address 2 different but related objectives: identifying the factors that contribute to the MWL of HCPs when using DHT and examining and exploring the applied assessments for the measurement of MWL with a special focus on eye tracking. Methods Following the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) 2020 statement, we conducted a systematic review and processed a literature search in the following databases: MEDLINE (PubMed), Web of Science, Academic Search Premier and CINAHL (EBSCO), and PsycINFO. Studies were eligible if they assessed the MWL of HCPs related to DHT. The review was conducted as per the following steps: literature search, article selection, data extraction, quality assessment (using the Standard Quality Assessment Criteria for Evaluation Primary Research Papers From a Variety of Fields [QualSyst]), data analysis, and data synthesis (narrative and tabular). The process was performed by 2 reviewers (in cases of disagreement, a third reviewer was involved). Results The literature search process resulted in 25 studies that fit the inclusion criteria and examined the MWL of health care workers resulting from the use of DHT in health care settings. Most studies had sample sizes of 10-50 participants, were conducted in the laboratory, and had quasi-experimental or cross-sectional designs. The main results can be grouped into two categories: assessment methods and factors related to DHT that contribute to MWL. Most studies applied subjective methods for the assessment of MWL. Eye tracking did not play a major role in the selected studies. The factors contributing to a higher MWL were clustered into organizational and systemic factors. Conclusions Our review of 25 papers shows a diverse assessment approach toward the MWL of HCPs related to DHT as well as 2 groups of relevant contributing factors to MWL. Our results are limited in terms of interpretability and causality due to methodological weaknesses of the included studies and may be limited by some shortcomings in the search process. Future research should concentrate on adequate assessments of the MWL of HCPs dependent on the setting, the evaluation of quality criteria, and further assessment of the contributing factors to MWL. Trial Registration PROSPERO (International Prospective Register of Systematic Reviews) CRD42021233271; https://www.crd.york.ac.uk/PROSPERO/display_record.php?ID=CRD42021233271