Aim: An ultrasensitive nano UHPLC–ESI–MS/MS method is developed to simultaneously monitor three low-concentration neuromedin-like peptides in microdialysates. Results: Peptide preconcentration and sample desalting is performed online on a trap column. A shallow gradient slope at 300 nl/min on the analytical column maintained at 35°C, followed by two saw-tooth column wash cycles, results in the highest sensitivity and the lowest carryover. The validated method allows the accurate and precise quantification of 0.5 pM neurotensin and neuromedin N (2.5 amol on column), and of 3.0 pM neuromedin B (15.0 amol on column) in in vivo microdialysates without the use of internal standards. Conclusion: The assay is an important tool for elucidating the role of these neuromedin-like peptides in the pathophysiology of neurological disorders.