<p><i>Uncaria tomentosa</i> (Rubiaceae) is a medicinal plant that is widely recognized for its potent anti-inflammatory activity. Chlorogenic acid derivatives, also called carboxyl alkyl esters (CAEs), are a class of compounds produced by <i>Uncaria</i> species that are well known for their anti-inflammatory properties. This work aimed to produce adventitious roots capable of producing CAEs, chlorogenic acid (<b>1</b>) and 3,4-di-O-caffeoylquinic acid (<b>2</b>), and to evaluate the cytotoxic potential of these compounds in order to ensure the safety of the crude extract obtained on the 35th day of culture. <i>Uncaria tomentosa</i> root cultures were established in Murashige &amp; Skoog medium supplemented with 3% (w/v) sucrose and 1 mg.L^− 1 1-naphthaleneacetic acid, micropropagated, and subcultured every 49 days. The root cultures produced 58.47 mg.g^− 1 dry weight (DW) of <b>1</b> and peak accumulation was observed at 35 days of culture, coinciding with the increase in biomass. Compound <b>2</b> of high pharmacological and commercial value also accumulated, reaching 32.53 mg.g^− 1 DW on day 42. Additionally, the cytotoxicity of the crude extract obtained on day 35 (containing 58.47 mg.g^− 1 DW of <b>1</b> and 20.28 mg.g^− 1 DW of <b>2</b>) was evaluated in mouse embryonic fibroblasts (NIH/3T3) and murine melanoma cells (B16-F10). The extract exhibited no cytotoxic effects in vitro, supporting its safety profile in prospective applications. These findings indicate substantial progress toward the large-scale in vitro culture of adventitious roots, a pivotal step in the development of anti-inflammatory herbal medicines derived from <i>U. tomentosa</i> tissue cultures.</p>