Effective implementation and sustainability of quality laboratory programmes in Sub-Saharan Africa relies on the development of appropriate staff retention strategies. Assessing the factors responsible for job satisfaction and retention is key for tailoring specific interventions aiming at improving the overall impact of health programmes. A survey was developed to assess these factors among 224 laboratorians working in the laboratory programme the University of Maryland implemented in seven Sub-Saharan African countries. Lack of professional development was the major reason for leaving the previous job for 28% of interviewees who changed jobs in the past five years. Professional development/training opportunities was indicated by almost 90% (195/224) of total interviewees as the most important or a very important factor for satisfaction at their current job. Similarly, regular professional development/opportunities for training was the highest rated incentive to remain at their current job by 80% (179/224). Laboratory professionals employed in the private sector were more likely to change jobs than those working in the public sector (P = 0.002). The findings were used for developing specific strategies for human resources management, in particular targeting professional development, aiming at improving laboratory professionals within the University of Maryland laboratory programme and hence its long-term sustainability.