In multi-analyte procedures, sufficient separation is important to avoid interferences, particularly when using liquid chromatography/mass spectrometry (LC/MS) because of possible ion suppression or enhancement. However, even using ultra-high-performance LC, baseline separation is not always possible. For development and validation of an LC/MS/MS approach for quantification of 140 antidepressants, benzodiazepines, neuroleptics, beta-blockers, oral antidiabetics, and analytes measured in the context of brain death diagnosis in plasma, the extent of ion suppression or enhancement of co-eluting analytes within and between the drug classes was investigated using atmospheric-pressure chemical ionization (APCI) or electrospray ionization (ESI). Within the drug classes, five analytes showed ion enhancement of over 25% and six analytes ion suppression of over 25% using APCI and 16 analytes ion suppression of over 25% using ESI. Between the drug classes, two analytes showed ion suppression of over 25% using APCI. Using ESI, one analyte showed ion enhancement of over 25% and five analytes ion suppression of over 25%. These effects may influence the drug quantification using calibrators made in presence of overlapping and thus interfering analytes. Ion suppression/enhancement effects induced by co-eluting drugs of different classes present in the patient sample may also lead to false measurements using class-specific calibrators made in absence of overlapping and thus interfering analytes. In conclusion, ion suppression and enhancement tests are essential during method development and validation in LC/MS/MS multi-analyte procedures, with special regards to co-eluting analytes.