ABSTRACT While advances in nucleic acid sequencing to achieve parallel and single molecule analysis have been astounding, analogous techniques for peptide/protein sequencing are lacking. Tandem mass-spec analysis still requires millions of copies of a protein and is inherently serial rather than parallel. Thus, the current approaches being applied to parallel and single molecule protein analysis primarily focus on the use of modified nanopores. But their ability to differentiate subtle differences between individual amino acids (AAs), or strings of AAs, only works in isolated cases. To create a parallel single molecule sequencing platform for peptides/proteins, in collaboration with the Marcotte group, we devised a technique called fluorosequencing. In this technique, we label AAs with fluorophores, and on a TIRF 4-channel microscope the N-terminal AAs are removed using classic Edman degradation. As fluorophore labelled AAs are iteratively removed, both their identity and position are revealed, generating a partial-sequence. The partial-sequences are compared to a genomic database of all possible proteins, thereby revealing the proteins in the sample. Mixtures of millions of peptides are analyzed in parallel; single molecules at a time. Albeit the method is functional, there are several aspects that require improvement to generate a mature technology. One obstacle that we will overcome is the general lack of approaches that allow sequential and selectively labelling of multiple amino acids on the same peptide/protein with different tags, as well as differentiating the N-terminal and C-terminal residues from lysine and Glu/Asp AAs, respectively. We will also explore labeling four AA within the following set: Cys, Lys, Tyr, Typ, His, Ser, Thr, Glu/Asp, Arg, PSer, PThr, PTyr (P = phospho). Moreover, we feel that fluorosequencing can be readily extended to post-translational modifications (PTMs) such as mono, di, and trimethylated Lys, as well as ubiquitination. The seq