D. Tech. (Department Logistics, Faculty of Management Sciences), Vaal University of Technology / The majority of business incubators in South Africa are supported by the Small Enterprise Development Agency (SEDA). However, a notable proportion of business incubators face a number of challenges to growth and development. Despite a number of studies conducted on business incubators generally, little information exists on the framework for the creation of business incubators. The concept of business incubation has gained prominence in academia in recent years as a vehicle for small business development. In view of the growing importance of business incubators, the objective of this study is to develop a framework for the creation of business incubators in South Africa. Business incubator managers, coaches, project specialists, and enterprise development practitioners were selected, being considered the ideal target population for this study. The study adopted a sequential mixed-methods methodology that commenced with a qualitative study and was followed by a quantitative study. For the qualitative study, data were collected from a purposively selected business incubator sample comprising nine participants. The principle of technical saturation was applied to ascertain the adequacy of the sample size. The trustworthiness and credibility of the qualitative study were achieved through pre-testing of the interview guide, bracketing, prolonged engagement with participants, peer debriefing, and researcher reflexivity. The analysis of the qualitative data was conducted using content and thematic analyses. The qualitative element of the study identified incubator prerequisites, situational analyses, operational processes/incubation strategies, and operational outputs as the main determinants of the framework for business incubators. In line with the methodology of the study, the determinants of the framework for business incubators that emerged from the qualitative study were further examined through a quantitative study. The data for the quantitative study were generated from a conveniently selected incubator practitioner sample of 121 respondents, using a structured self-administered questionnaire. Furthermore, the historical evidence method guided the determination of the sample size for the quantitative study. Prior to questionnaire administration of this study a pilot study was conducted to improve the accuracy of the survey instrument. The quantitative section utilised statistical data analysis procedures, descriptive statistics, reliability and validity analysis, correlation analysis and confirmatory factor analysis. The Statistical Package for Social Sciences (SPSS) software was utilised to analyse the quantitative data. The results of the quantitative data analysis revealed that the framework for incubation presents incubator prerequisites (incubation entry of incubatees) as the predictor construct. This construct has eight sub-components, which are situational analysis (incubatees’ characteristics), key requirements, operational process, factor components, intellective capabilities, administrative capabilities, market force engineering, and strategic resourcing (impact) with KPIs. The mediating construct is business incubator performance (incubation process—industry coaches, governance structure, and entrepreneurial focus), which in turn leads to monitoring and evaluation (incubation output—graduation phase, impact, and follow-ups). The relationships between these constructs were tested. In order to verify the reliability of the measurement items of the current study, Cronbach’s alpha coefficient, the item-to-total values and composite reliability were computed. The validity of the survey instrument was enhanced through content, convergent, discriminant and predictive validities. In addition, the reliability and validity measures employed in the present study showed that the survey instrument utilised in the quantitative study was both reliable and valid. The study is significant in terms of its theoretical and business incubation policy implications. Theoretically, the study provides a comparative impression of the South African business incubation perspective and current trends in the ecosystem. Thus, future researchers, particularly in developing countries, may use the results of this study as a reference benchmark in terms of literature and research methodology. The various organs of the state, government agencies, as well as non-governmental organisations that are responsible for entrepreneurship development and the creation of additional business incubators, may use the study as a reference point in the generation of different initiatives aimed at improving the small business environment in South Africa.