Since its development in 1993, the Trier Social Stress Test (TSST) has been used widely as a psychosocial stress paradigm to activate the sympathetic nervous system and hypothalamic-pituitary-adrenal axis (HPAA) stress systems, stimulating physiological functions (e.g. heart rate) and cortisol secretion. Several methodological variations introduced over the years have led the scientific community to question replication between studies. In this systematic review, we used the Preferred Reporting Items of Systematic Reviews and Meta-Analysis (PRISMA) to synthesize procedure-related data available about the TSST protocol to highlight commonalities and differences across studies. We noted significant discrepancies across studies in how researchers applied the TSST protocol. In particular, we highlight variations in testing procedures (e.g., number of judges, initial number in the arithmetic task, time of the collected saliva samples for cortisol) and discuss possible misinterpretation in comparing findings from studies failing to control for variables or using a modified version from the original protocol. Further, we recommend that researchers use a standardized background questionnaire when using the TSST to identify factors that may influence physiological measurements in tandem with a summary of this review as a protocol guide. More systematic implementation and detailed reporting of TSST methodology will promote study replication, optimize comparison of findings, and foster an informed understanding of factors affecting responses to social stressors in healthy people and those with pathological conditions.