Introduction: Balamuthia mandrillaris is an opportunistic free-living amoeba that causes granulomatous amoebic encephalitis, a rare but often fatal disease with delayed diagnosis. Laboratory identification is challenging because of low organism burden, slow growth, and morphological similarity to other free-living amoebae. Consequently, several laboratory techniques have been developed for detection, isolation, or cultivation, though their performance and feasibility vary across studies. This systematic review evaluates and compares these methods. Methods: Following PRISMA 2020 guidelines, a systematic search (24/09/2025) was conducted in Medline, Web of Science, ScienceDirect, and ProQuest using “Balamuthia mandrillaris” combined with “Detection”, “Isolation”, “Culture Techniques”, and “In Vitro Techniques”. Experimental and observational laboratory studies assessing detection, isolation, or cultivation methods in clinical, veterinary, or environmental samples were included. Outcomes comprised methodological performance, feasibility, and cultivation success. Results: Of 350 articles screened, 10 studies met the inclusion criteria. Molecular methods, particularly PCR, demonstrated the highest analytical performance, with sensitivities up to 84% in formalin-fixed tissues and limits of detection as low as ~1 amoeba per sample. In environmental studies, PCR showed higher detection rates than culture (e.g., 47.4% vs 18.4% for free-living amoebae). Culture-based methods enabled isolation but required prolonged incubation (up to 60 days) and showed low recovery (e.g., 4.5% in water samples). Multi-step protocols combining environmental culture, mammalian cell co-culture, and axenization improved isolation but remained technically demanding. In vitro cultivation advances demonstrated sustained growth in human fibroblast and endothelial cell lines and axenic media, achieving yields up to 1.3 × 10⁶ amoebae/mL with long-term viability. Immunological assays were useful for exposure assessment but insufficient as standalone diagnostics. Conclusions: Molecular methods provide the most sensitive detection, while culture remains essential for isolation despite low efficiency. Integrated workflows and standardized protocols are needed to improve diagnostic reliability and support future research.