Short open reading frame (sORF)-encoded peptides (sPEPs) have been found across a wide range of genomic locations in a variety of species. To date, their identification, validation, and characterisation in the human fungal pathogen Cryptococcus neoformans has been limited due to a lack of standardised protocols. We have developed an enrichment process that enables sPEP detection within a protein sample from this polysaccharide-encapsulated yeast, and implemented proteogenomics to provide insights into the validity of predicted and hypothetical sORFs annotated in the C. neoformans genome. Novel sORFs were discovered within the 5’ and 3’ UTRs of known transcripts and non-coding RNAs. One novel candidate, dubbed NPB1, that resided in an RNA annotated as “non-coding” was chosen for characterisation. Through the creation of both specific point mutations and a full deletion allele with the aid of the newly developed amdS2 Blaster for C. neoformans, the function of the new sPEP, Npb1, was shown to resemble that of the bacterial trans-translation protein SmpB.