The 5-hydroxytryptamine 1A receptor (serotonin 1A receptor; 5-HT1AR) is involved in a large series of brain functions, and roles in anxiety, depression, and cognition have been reported. So far, published information on mass spectrometrical characterization of 5-HT1AR is limited to the presence of two 5-HT1AR peptides in rat's whole brain as observed by in-solution digestion followed by LC-MS/MS. Knowledge about the protein sequence and PTMs, however, would have implications for generation of specific antibodies and designing studies on the 5-HT1AR at the protein level. A rat recombinant 5-HT1AR was extracted from the tsA201 cell line, run using several gel-based principles with subsequent in-gel digestion with several proteases, chymotrypsin, trypsin, AspN, proteinase K, and pepsin followed by nano-LC-ESI-MS/MS analysis on a high capacity ion trap and an LTQ Orbitrap Velos. Using two search engines, Mascot and Modiro™, the recombinant 5-HT1AR was identified showing 94.55% sequence coverage. A single phosphorylation at S301 was identified and verified by phosphatase treatment and a series of amino acid substitutions were detected. Characterization of 5-HT1AR, a key player of brain functions and neurotransmission, was shown and may enable generation of specific antibodies, design of future, and interpretation of previous studies in the rat at the protein level.