The analysis of volatile organic compounds (VOCs) within breath for noninvasive disease detection and monitoring is an emergent research field that has the potential to reshape current clinical practice. However, adoption of breath testing has been limited by a lack of standardization. This protocol provides a comprehensive workflow for online and offline breath analysis using selected ion flow tube mass spectrometry (SIFT-MS). Following the suggested protocol, 50 human breath samples can be analyzed and interpreted in <3 h. Key advantages of SIFT-MS are exploited, including the acquisition of real-time results and direct compound quantification without need for calibration curves. The protocol includes details of methods developed for targeted analysis of disease-specific VOCs, specifically short-chain fatty acids, aldehydes, phenols, alcohols and alkanes. A procedure to make custom breath collection bags is also described. This standardized protocol for VOC analysis using SIFT-MS is intended to provide a basis for wider application and the use of breath analysis in clinical studies.