In forensic bioanalytical methods, there is a general agreement that calibrators should be prepared by fortifying analytes in matrix-based blank samples (matrix-based). However, in the case of vitreous humor (VH), the collection of blank samples for the validation and for routine analysis would require the availability of many cadavers. Besides the difficulty of obtaining enough blank VH, this procedure could also represent an ethical issue. Here, a study of matrix effect was performed taking into consideration human and bovine vitreous and saline solution (SS) (NaCl 0.9%). Tricyclic antidepressants [amitriptyline (AMI), nortriptyline (NTR), imipramine (IMI) and desipramine (DES)] were used as model analytes and were extracted from samples by means of liquid-phase microextraction and detected by gas chromatography-mass spectrometry. Samples of human and bovine VH and SS were prepared in six different concentrations of antidepressants (5, 40, 80, 120, 160 and 200 ng/mL) and were analyzed. Relative matrix effect was evaluated by applying a two-tailed homoscedastic Student's t-test, comparing the results obtained with the set of data obtained with human VH and bovine VH and SS. No significant matrix effect was found for AMI and NTR in the three evaluated matrices. However, a great variability was observed for IMI and DES for all matrices. Once compatibilities among the matrices were demonstrated, the method was fully validated for AMI and NTR in SS. The method was applied to six VH samples deriving from real cases whose femoral whole blood (FWB) was analyzed by a previously published method. An average ratio (VH/FWB) of ∼ 0.1 was found for both compounds.