This study investigates the impact of TDP-43 dysfunction on cryptic exon (CE) splicing and its consequences for protein function in ALS/FTD. TDP-43, an RNA-binding protein, is genetically and pathologically linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Loss of TDP-43 function leads to the inclusion of cryptic exons in hundreds of transcripts, which can alter cellular function through loss-of-function mechanisms. The study demonstrates that mRNA transcripts harboring cryptic exons generate de novo proteins in TDP-43-depleted human iPSC-derived neurons in vitro, and these de novo peptides are also present in cerebrospinal fluid (CSF) samples from ALS/FTD patients. Using transcriptomic and proteomic analyses, 65 peptides were identified that map to 12 CEs. These CEs were predictive of those expressed in postmortem brain tissue from ALS/FTD patients. The study also shows that the inclusion of cryptic peptide sequences alters protein interactions, potentially altering their function. Finally, a targeted proteomics assay was developed to identify 18 de novo "cryptic" peptides across 13 genes in CSF samples from ALS/FTD patients. These findings suggest new mechanisms for ALS/FTD pathophysiology downstream of TDP-43 dysfunction and provide a potential strategy to assay TDP-43 function in patient CSF.This study investigates the impact of TDP-43 dysfunction on cryptic exon (CE) splicing and its consequences for protein function in ALS/FTD. TDP-43, an RNA-binding protein, is genetically and pathologically linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Loss of TDP-43 function leads to the inclusion of cryptic exons in hundreds of transcripts, which can alter cellular function through loss-of-function mechanisms. The study demonstrates that mRNA transcripts harboring cryptic exons generate de novo proteins in TDP-43-depleted human iPSC-derived neurons in vitro, and these de novo peptides are also present in cerebrospinal fluid (CSF) samples from ALS/FTD patients. Using transcriptomic and proteomic analyses, 65 peptides were identified that map to 12 CEs. These CEs were predictive of those expressed in postmortem brain tissue from ALS/FTD patients. The study also shows that the inclusion of cryptic peptide sequences alters protein interactions, potentially altering their function. Finally, a targeted proteomics assay was developed to identify 18 de novo "cryptic" peptides across 13 genes in CSF samples from ALS/FTD patients. These findings suggest new mechanisms for ALS/FTD pathophysiology downstream of TDP-43 dysfunction and provide a potential strategy to assay TDP-43 function in patient CSF.