An important safety consideration for topically administered drugs is the extent of systemic exposure they achieve. The aim of this study was to evaluate whether a topically administered microdose of the model drug diclofenac can predict the systemic availability, plasma, and tissue pharmacokinetics of a topical therapeutic dose. Eight healthy participants (6 men and 2 women) participated in a 4-period, crossover study. In period 1, a topical microdose (62 ± 6 μg) was administered; in period 2, a single intravenous microdose (0.95 ± 0.03 μg) was administered; in period 3, a topical therapeutic dose (120 mg) was administered; and in period 4, a single intravenous therapeutic dose (75 mg) of [C]diclofenac was administered, with or without the addition of unlabeled diclofenac. Venous blood, urine, and microdialysis samples from subcutaneous adipose tissue beneath the dermal application site were collected for 24 h post-dosing. Total C-concentrations in plasma and microdialysates were quantified using accelerator mass spectrometry. The disposition of intravenously administered [C]diclofenac was dose-linear. However, after topical administration, the fraction of total C absorbed (geometric mean and 95% confidence interval) was higher (P = .0019, 2-tailed, paired t test) for the microdose (0.48% and 0.34%-0.67%) compared with the therapeutic dose (0.13% and 0.07%-0.22%) (geometric mean ratio and 90% confidence interval: 3.79 and 2.41-5.98). Dose-normalized C-concentrations in microdialysates were low, variable, and did not differ between doses. Our study demonstrates the feasibility of quantifying C-concentrations in plasma and microdialysates following the topical administration of a microdose of [C]diclofenac. The observed nonlinearity in systemic availability after topical dosing suggests that microdosing may not accurately predict the disposition of certain topical drugs at therapeutic doses. SIGNIFICANCE STATEMENT: We assessed whether a topically applied microdose of diclofenac could predict the systemic availability of a therapeutic dose. Results showed that systemic absorption was not dose-linear, indicating that microdosing may have limited use for predicting the pharmacokinetics of some topical drugs.