Several studies investigated diclofenac tissue concentrations using microdialysis (MD). However, thorough evaluations of the optimal MD set-up for diclofenac are unavailable. Thus, this in vitro MD study aimed to compare different set-ups to improve quantitative recovery of diclofenac. In forward and reverse in vitro MD experiments with diclofenac at two concentrations (1 and 100 ng/ml), the perfusion solutions physiological saline 0.9% (PS) and human albumin 1% (HSA) were compared using tissue probes (10-mm membrane) and customized intravenous (iv) probes (30-mm membrane). Using PS, the mean relative recovery of diclofenac at 1 ng/ml was 1.6% ± 0.04% and 3.12% ± 0.00% with the tissue probe and the iv probe, respectively. The respective mean relative recovery for diclofenac at 100 ng/ml was 0.02% ± 0.01% and 0.21% ± 0.11%. Using HSA, the mean relative recovery was 314% ± 25% (tissue probe) and 1064% ± 97% (iv probe) for diclofenac at 1 ng/ml and 444% ± 91% and 1415% ± 217% for diclofenac at 100 ng/ml. In reverse dialysis using PS, the mean relative loss of diclofenac was 99.2% ± 0.5% (tissue probe) and 95.8% ± 1.7% (iv probe). Using HSA, the mean relative loss was -4.4% ± 7.2% and 0.2% ± 7.5%, respectively. PS and HSA were not suitable perfusion solutions for quantification of absolute diclofenac concentrations. Despite methodological challenges, HSA may be used for comparative experiments or bioequivalence studies.