The optical response of four new anthracenylmethyl pendant-arm derivatives (L1-L4) of the macrocyclic ligands aneNS3, aneNS 2O, aneNS2O2, and aneN2SO toward the metal ions Zn2+, Cd2+, Pb2+, Cu 2+, Hg2+, Ag+, Fe2+, Co 2+, Ni2+, Mn2+, Ca2+, Na +, Mg2+, and K+ was investigated in 1:1 (v/v) MeCN/H2O solutions. A strong chelation enhancement of quenching effect was observed on the fluorescent emission intensity of L2 as a consequence of the host-guest interaction with Hg2+ and the formation of a 1:2 metal-to-ligand complex. Density functional theory calculations confirmed the formation of a sandwich-type complex between L2 and Hg2+ as a favorable process. A matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry study using the four ligands as active MALDI probes was also performed. L1-L4 have also been explored as fluorescence chemosensors in microsamples using NANODROP technology.