Peroxisome biogenesis disorders (PBDs) are a group of autosomal-recessive developmental and progressive metabolic diseases leading to the Zellweger spectrum (ZS) phenotype in most instances. Diagnosis of clinically suspected cases can be difficult because of extensive genetic heterogeneity and large spectrum of disease severity. Furthermore, a second group of peroxisomal diseases caused by deficiencies of single peroxisomal enzymes can show an indistinguishable clinical phenotype. The diagnosis of these peroxisomal disorders relies on the clinical presentation, the biochemical parameters in plasma and erythrocyte membranes, and genetic testing as the final step. Analysis of patients' cells is frequently required during the diagnostic process, e.g., for complementation analysis to identify the affected gene before sequencing. In the cases with unclear clinical or biochemical presentation, patients' cells are analyzed to prove PBD or to demonstrate biochemical abnormalities that might be elusive in plasma. Cell lines from skin fibroblast that are usually generated for diagnostic workup are not available in all instances, mainly because the required skin biopsy is invasive and sometimes denied by parents. An alternative cellular system has not been analyzed sufficiently. In this study, we evaluated the alternative use of lymphoblastoid cell lines (LCLs), derived from a peripheral blood sample, in the diagnostic process for PBD. LCLs were suitable for immunofluorescence visualization of peroxisomal enzymes, complementation analysis, and the biochemical analysis to differentiate between control and PBD LCL. LCLs are therefore an easily obtainable alternative cellular system for a detailed PBD diagnostic workup with a reliability of diagnostic results equal to those of skin fibroblasts.