A simple, fast, and cost-effective organic solvent-based protocol was developed for DNA extraction from deer antlers and prepared trophy skulls without the need for commercial kits or cryogenic grinding. The method combines bead-based mechanical homogenization with a 4-hour enzymatic digestion in EDTA buffer containing N-lauryl sarcosine and Proteinase K, followed by phenol-chloroform-isoamyl alcohol purification and centrifugal filtration. DNA quality and quantity were evaluated using agarose gel electrophoresis, Qubit fluorometry, and Nanodrop spectrophotometry. The protocol was tested on 60 samples, including 31 antlers and 29 pedicle parts of prepared trophy skulls from roe deer (Capreolus capreolus), fallow deer (Dama dama), and red deer (Cervus elaphus). Statistical analysis was performed to assess concentration differences among species and sample types. To assess suitability for downstream applications, species-specific microsatellite markers were amplified using multiplex PCR, successfully generating complete genotypes from all 60 samples. These results, along with a demonstrated case study, confirm that the developed protocol provides high-quality DNA suitable for molecular genetic investigations, enabling reliable genotyping from small amounts of both antler and processed trophy materials in forensic and conservation contexts.