Sperm separation through protein activation or inhibition, based on proteins differentially expressed in X- and Y-chromosome-bearing sperm, represents a novel approach to controlling offspring sex. This method separates X- and Y-bearing sperm by directly activating these differentially expressed proteins. However, technical limitations and inconsistent sperm concentrations remain. This study established a Toll-like receptors 7 and 8 (TLR7/8) activation-based sorting system for goat sperm and developed a corresponding technical protocol. Building on our laboratory's previous technique for sorting X- and Y-chromosome-bearing sperm by targeting specific proteins, we investigated interactions among sperm concentration, agonist R848 concentration, and incubation time. R848 (resiquimod) is a synthetic imidazoquinoline compound that acts as an agonist of Toll-like receptors 7 and 8 (TLR7/8). The results indicated that R848 binding to TLR7/8 on the tails of X-bearing sperm inhibited their motility, causing them to accumulate in the lower fraction of the sorting system. In contrast, Y-bearing sperm motility was unaffected, allowing these sperm to migrate to the upper fraction. Acrosome integrity did not differ significantly between the selected experimental groups and the control group, with values exceeding 50%. Plasma membrane integrity in the selected experimental groups was either significantly higher than or not significantly different from that in the control group. SRY immunofluorescence staining revealed X-bearing sperm proportions of 88.29% ± 1.68%, 84.50% ± 0.18%, and 80.32% ± 1.94% in one set of selected experimental groups, and Y-bearing sperm proportions of 80.95% ± 0.00%, 88.89% ± 0.00%, and 84.03% ± 2.38%. We subsequently investigated the effect of centrifugal force on R848 removal. The results demonstrated that centrifugation effectively removed R848. Only in this study, the optimal centrifugal force was 800 × g for sorted X-bearing sperm and 700 × g for sorted Y-bearing sperm from dairy goats. Finally, the optimized sorting system was used for sperm separation. In a single independent exploratory IVF batch, 21 of 25 sexed embryos in the upper-fraction B7 group were classified as male (84.0%), whereas 16 of 20 sexed embryos in the lower-fraction E8 group were classified as female (80.0%). B7 and E8 were intended for Y- and X-bearing sperm enrichment, respectively. These observations were limited to the embryos analyzed in this batch and do not establish the reproducibility of sperm enrichment or the preservation of fertilization and developmental competence. This study demonstrated that the interaction among goat sperm concentration, R848 concentration, and incubation time significantly affected target sperm enrichment, while adjusting centrifugal force improved the efficiency of R848 removal. Specifically, the optimized protein-targeted system for sorting X-bearing sperm from dairy goats used a sperm concentration of 6 × 108 cells/mL and 1.4 μmol/L R848, with two incubation steps totaling 30 min. The lower sperm fraction was collected and washed twice by centrifugation at 800×g. The system for sorting Y-bearing sperm used a sperm concentration of 6 × 108 cells/mL and 0.6 μmol/L R848, with two incubation steps totaling 45 min. The upper sperm fraction was collected and washed twice by centrifugation at 700×g.