Homosalate (HMS) is an organic UV filter used in sunscreens and personal care products. Despite its widespread use and detection in environmental matrices, little is known regarding its exposure in humans. HMS is used as a mixture of cis- and trans-isomers, and we recently revealed major differences in human toxicokinetics, indicating the need to consider these isomers separately in exposure and risk assessments. In the course of these previous investigations of human HMS toxicokinetics, we identified two trans-HMS-specific and one cis-HMS-specific biomarker candidates. However, the latter lacks sensitivity due to only low amounts excreted in urine, prompting the search for another cis-HMS-specific biomarker. Our toxicokinetic investigations revealed a total of five isomers of HMS carboxylic acid metabolites (HMS-CA). Of these, only one was specifically formed from cis-HMS (HMS-CA 5), but its full identity in terms of constitution and configuration had, so far, not been elucidated. Here, we describe the synthesis of three HMS-CA isomers, of which the isomer (1R,3S,5S)/(1S,3R,5R)-3-((2-hydroxybenzoyl)oxy)-1,5-dimethylcyclohexane-1-carboxylic acid turned out to be HMS-CA 5. Taken together with two previously synthesized HMS-CA isomers, we were able to identify the constitution and configuration of all five HMS-CA isomers observed in human metabolism. We integrated the newly identified cis-HMS-specific metabolite HMS-CA 5 into our previously published human biomonitoring LC-MS/MS method. Intra- and interday precisions had coefficients of variation below 2% and 5%, respectively, and the mean relative recovery was 96%. The limit of quantification in urine was 0.02 μg L-1, enabling the quantification of HMS-CA 5 in urine samples for at least 96 h after sunscreen application. The extended method thus enables the sensitive and separate monitoring of cis- and trans-HMS in future human biomonitoring studies for exposure and risk assessment.