CDK5 is a key regulator of neuronal homeostasis, and its dysregulation has been associated with altered secretory pathways. Here, we investigated how CDK5 knockout (KO) affects the neuronal secretome using FN2.1 human induced pluripotent stem cells (hiPSC)-derived neurons. Both wild-type (WT) and CDK5-KO FN2.1 lines were successfully differentiated, expressing the neuronal markers TUJ-1, MAP2, and MAP5. Conditioned media collected after 18 h of starvation were analyzed by MS/MS proteomics, and 48 differentially expressed proteins were identified using the Limma package in R. Gene Ontology analysis revealed enrichment in processes related to focal adhesion and actin cytoskeleton regulation. Consistently, cell-attachment assays demonstrated a significant reduction in adhesion of CDK5-KO FN2.1-derived neurons. Notably, the CDK5-KO secretome showed significantly increased levels of MAPT (TAU) and TDP-43, which were further confirmed in total cell lysates by Western blot. Together, these findings suggest that CDK5 deficiency reshapes the neuronal secretory profile, weakens cell adhesion, and increases the levels of TAU and TDP-43, ultimately disturbing neuronal homeostasis.