Ablative fractional laser-assisted drug delivery has gained attention as a promising method for enhancing dermal drug absorption and improving therapeutic outcomes in dermatological conditions, particularly for hypertrophic and keloid scars. However, despite the growing number of clinical trials and case reports supporting its efficacy, there remains a scarcity of robust evidence on the topical bioavailability and dermato-pharmacokinetics of drugs in human subjects. This study aimed to examine the enhancement of triamcinolone acetonide (TAC) bioavailability following treatment with a fractional Erbium-Doped Yttrium Aluminum Garnet (Er: YAG) laser. Stratum corneum (SC) uptake and transport of TAC from 0.1% TAC cream and 10 mg/mL TAC solution/suspension with and without the laser pre-treatment were determined through tape stripping method for SC collection. TAC therein was quantified by an ultra-performance liquid chromatography coupled with photodiode array (UPLC-PDA) detection. TAC from both formulations without laser assistance was percutaneously absorbed within 6 h and TAC was delivered out from the solution to the SC remarkably higher. When the skin was pre-treated with the laser, permeability of TAC from the solution was escalated by 5 folds. TAC distribution profiles in the SC also confirmed this increased drug uptake, mainly the outer skin layers. On the other hand, amounts of absorbed TAC and their distribution patterns from the cream remained unchanged and low. No adverse events and unbearable pain were observed throughout the experiments. The fractional Er: YAG laser enhanced the dermal absorption of TAC, but this effect was confined to the solution formulation, with no significant improvement seen in the cream. This finding highlights the critical role that drug formulation plays in laser-assisted drug delivery. Moreover, factors such as drug selection, laser type, and optimal laser settings may also impact the efficacy of this approach and require further exploration.