Background/purpose: Foot malodor is mostly due to short-chain fatty acids produced by bacterial metabolism of eccrine sweating. We aimed to develop a protocol for an objective (instrumental) efficacy evaluation of foot deodorant formulations. Methods: Head-space solid-phase microextractions of target fatty acids from the feet of six healthy volunteers were analyzed by GC-MS. A comparative analysis of the treated vs. the untreated foot was performed in each subject after washing the feet with a physiologic solution and incubating at 36 °C for 24–72 h in tryptic soy agar growth medium. Results: Acetic, butyric, isobutyric and isovaleric acids were identified as the main contributors to foot malodor in the majority of volunteers. Propionic, valeric and isocaproic acids were also detected in some subjects. Comparative analysis according to the protocol developed showed a statistically significant (P<0.01) reduction of target fatty acids ranging from −26.6% to −77.0%. Conclusion: The protocol developed is a convenient, sensitive and non-invasive method to test the efficacy of foot deodorant formulations in human volunteers.